Zea mays regulatory elements and uses thereof

ABSTRACT

Provided are vector constructs and methods for expressing a transgene in plant cells and/or plant tissues using gene regulatory elements, including the promoters, 5′-UTRs, introns, and/or 3′-UTRs, isolated from  Zea mays.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit, under 35 U.S.C. 5 § 119(e), to U.S.Provisional Patent Application No. 61/890,904, filed Oct. 15, 2013, thecontents of which are incorporated by reference in their entirety intothe present application.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 8 KB ASCII (Text) file named “74276”created on Oct. 3, 2014.

FIELD OF THE INVENTION

This invention is generally related to the field of plant molecularbiology, and more specifically, to the field of stable expression ofgenes in transgenic plants.

BACKGROUND

Plant transformation is an attractive technology for use in introducingagronomically desirable traits or characteristics into different cropplant species. Plant species are developed and/or modified to haveparticular desirable traits. Generally, desirable traits include, forexample, improving nutritional value quality, increasing yield,conferring pest or insect resistance, disease resistance, increasingdrought and stress tolerance, improving horticultural qualities (e.g.,pigmentation and growth), imparting herbicide tolerance, enabling theproduction of industrially useful compounds and/or materials from theplant, and/or enabling the production of pharmaceuticals.

Transgenic plants comprising multiple transgenes stacked at a singlegenomic locus are produced via plant transformation technologies. Planttransformation technologies confer the introduction of transgenes into aplant cell, recovery of a fertile transgenic plant that contains thestably integrated copy of the transgene in the plant genome, andsubsequent transgene expression via transcription and translation of thetransgene(s). Thereby resulting in transgenic plants that possessdesirable traits and phenotypes. Each transgene in a stack typicallyrequires an independent promoter for gene expression within a plant, andthus multiple promoters are used in a transgene stack.

The need for co-expression of multiple transgenes for regulating thesame trait frequently results in the repeated use of the same promoterto drive expression of the multiple transgenes. However, the repeateduse of promoters comprising sequences that share a high level ofsequence identity may lead to homology-based gene silencing (HBGS). HBGShas been observed to occur frequently in transgenic plants (Peremarti etal., 2010) when repetitive DNA sequences are used within a transgene. Inaddition, repeated use of similar DNA sequences in transgene constructshas proven to be challenging in Agrobacterium due to recombination andinstability of the plasmid.

Described herein are maize gene regulatory elements (e.g., promoter,5′-UTR, and 3′-UTR). Further described are constructs and methodsutilizing maize regulatory elements.

SUMMARY

Disclosed herein are purified polynucleotides, vectors, constructs andmethods for expressing a transgene in plant cells and/or plant tissues.In one embodiment, regulatory elements of a chlorophyll a/b gene arepurified from the Zea mays genomes and recombined with sequences notnatively linked to said regulatory elements to create an expressionvector for expressing transgenes in plant cells not native to thechlorophyll a/b regulatory sequences. In one embodiment an expressionvector is provided wherein the regulatory elements of a chlorophyll a/bgene are operably linked to a polylinker sequence. Such an expressionvector eases the insertion of a gene, transgene, or gene cassette intothe vector in an operably linked state with the chlorophyll a/b generegulatory sequences.

In an embodiment, a construct is provided comprising a Zea mayschlorophyll a/b promoter of SEQ ID NO:1. In an embodiment, a geneexpression cassette is provided comprising a Zea mays chlorophyll a/bpromoter operably linked to a transgene. In an embodiment, a geneexpression cassette includes a Zea mays chlorophyll a/b 5′-UTR operablylinked to a transgene. In an embodiment, a gene expression cassetteincludes a Zea mays chlorophyll a/b 5′-UTR operably linked to apromoter. In an embodiment, a gene expression cassette includes a Zeamays chlorophyll a/b intron operably linked to a transgene. In anembodiment, a gene expression cassette includes a Zea mays chlorophylla/b intron operably linked to a promoter. In an embodiment, a constructincludes a gene expression cassette Zea mays chlorophyll a/b 3′-UTR. Inan embodiment, a gene expression cassette includes Zea mays chlorophylla/b 3′-UTR operably linked to a transgene. In an embodiment, a geneexpression cassette includes at least one, two, three, five, six, seven,eight, nine, ten, or more transgenes.

In an embodiment, a gene expression cassette includes independently a) aZea mays chlorophyll a/b promoter, b) a Zea mays chlorophyll a/b intron,c) a Zea mays chlorophyll a/b 5′-UTR, and d) a Zea mays chlorophyll a/b3′-UTR.

In accordance with one embodiment a nucleic acid vector is providedcomprising a promoter operably linked to a non-chlorophyll a/btransgene, wherein the promoter consists of SEQ ID NO:1 or a sequencehaving 90% sequence identity with SEQ ID NO:1. In a further embodimentthe nucleic acid vector comprises a gene cassette, wherein the genecassette comprises a promoter, a non-chlorophyll a/b transgene and a 3′untranslated region, wherein the promoter consists of SEQ ID NO:1operably linked to a first end of a transgene, wherein the second end ofthe transgene is operably linked to a 3′ untranslated sequenceconsisting of SEQ ID NO:6.

Methods of growing plants expressing a transgene using the Zea mayschlorophyll a/b promoters, 5′-UTRs, introns, and 3′-UTRs are disclosedherein. Methods of culturing plant tissues and cells expressing atransgene using the Zea mays chlorophyll a/b promoters, 5′-UTRs,introns, and 3′-UTRs are also disclosed herein. In an embodiment,methods as disclosed herein include tissue-specific gene expression inplant stem, leaf, cob, silk, kernel, stem, husk and pollen tissues.

In a further embodiment, a method of enhancing the over-expression of agene of interest contained within a second gene expression cassette isdisclosed herein.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided comprising a promoter operably linked to a non-chlorophylla/b transgene, wherein the promoter comprises SEQ ID NO:1 or SEQ IDNO:5. In accordance with one embodiment a plant or plant cell isprovided comprising SEQ ID NO:1 or SEQ ID NO:5, or a sequence that has90% sequence identity with SEQ ID NO:1 operably linked to a transgene.In one embodiment the plant is a corn variety. In one embodiment aplant, plant tissue, or plant cell is provided comprising a promoteroperably linked to a non-chlorophyll a/b transgene, wherein the promoterconsists of SEQ ID NO:1 or SEQ ID NO:5. In one embodiment a plant orplant cell is provided comprising a gene cassette, wherein the genecassette comprises a promoter operably linked to a transgene, furtherwherein the promoter consists SEQ ID NO:1 or SEQ ID NO:5. In a furtherembodiment the promoter is operably linked to a first end of atransgene, wherein the second end of the transgene is operably linked toa 3′ untranslated sequence consisting of SEQ ID NO:6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic flow chart displaying the process of identifyinghigh expressing genes in maize using a transcriptional profilingapproach with Next Generation Sequencing (NGS).

FIG. 2 shows the pDAB114411 vector plasmid map depicting a geneexpression cassette for expression of a cry34Ab1 reporter gene driven bythe Zea mays promoter of SEQ ID NO:1 and terminated by the StPinII 3′UTR regulatory element.

FIG. 3 shows the pDAB116038 vector plasmid map depicting a geneexpression cassette for expression of a phiYFP reporter gene driven bythe Zea mays promoter of SEQ ID NO:1 and terminated by the Zea mays3′-UTR of SEQ ID NO:4 regulatory elements.

FIG. 4 shows a vector plasmid map of the pDAB101556 control vectorcontaining a YFP reporter gene instead of the cry34Ab1 reporter genepresent in the test promoter construct, pDAB114411. The YFP geneexpression was driven by the Zea mays ubiquitin-1 (ZmUbi1) promoter andterminated by the Zea mays Per5 (ZmPer5) 3′-UTR.

FIG. 5 shows a vector plasmid map of pDAB108746, a positive controlvector containing the cry34Ab1 reporter gene driven by the ZmUbi1promoter and terminated by the StPinII 3′-UTR.

FIG. 6 shows a map of pDAB113121, a positive control vector. Thisplasmid contains a phiYFP reporter gene driven by the ZmUbi1 promoterand terminated by the StPinII 3′ UTR. The phiYFP gene contains anintron. The aad-1 gene cassette is identical in both pDAB113121 andpDAB116038.

DETAILED DESCRIPTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

As used herein, the term “backcrossing” refers to a process in which abreeder crosses hybrid progeny back to one of the parents, for example,a first generation hybrid F1 with one of the parental genotypes of theF1 hybrid.

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. A promoter may contain specific sequencesthat are recognized by transcription factors. These factors may bind toa promoter DNA sequence, which results in the recruitment of RNApolymerase. For purposes of defining the present invention, the promotersequence is bound at its 3′ terminus by the transcription initiationsite (i.e., ribosome binding site) and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

For the purposes of the present disclosure, a “gene”, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

As used herein a “transgene” is defined to be a nucleic acid sequencethat encodes a gene product, including for example, but not limited to,an mRNA. In one embodiment the transgene is an exogenous nucleic acid,where the transgene sequence has been introduced into a host cell bygenetic engineering (or the progeny thereof) where the transgene is notnormally found. In one example, a transgene encodes an industrially orpharmaceutically useful compound, or a gene encoding a desirableagricultural trait (e.g., an herbicide-resistance gene). In yet anotherexample, a transgene is an antisense nucleic acid sequence, whereinexpression of the antisense nucleic acid sequence inhibits expression ofa target nucleic acid sequence. In one embodiment the transgene is anendogenous nucleic acid, wherein additional genomic copies of theendogenous nucleic acid are desired, or a nucleic acid that is in theantisense orientation with respect to the sequence of a target nucleicacid in a host organism.

As used herein the term “non-chlorophyll a/b transgene” is any transgenethat encodes a protein with less than 90% sequence identity to theprotein encoded by the Zea may chlorophyll a/b coding sequence (SEQ IDNO:20).

“Gene expression” as defined herein is the conversion of theinformation, contained in a gene, into a gene product.

A “gene product” as defined herein is any product produced by the gene.For example the gene product can be the direct transcriptional productof a gene (e.g., mRNA, tRNA, rRNA, small RNA, antisense RNA, interferingRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation. Gene expression canbe influenced by external signals, for example, exposure of a cell,tissue, or organism to an agent that increases or decreases geneexpression. Expression of a gene can also be regulated anywhere in thepathway from DNA to RNA to protein. Regulation of gene expressionoccurs, for example, through controls acting on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization, or degradation of specific protein molecules afterthey have been made, or by combinations thereof. Gene expression can bemeasured at the RNA level or the protein level by any method known inthe art, including, without limitation, Northern blot, RT-PCR, Westernblot, ELISA assay, or in vitro, in situ, or in vivo protein activityassay(s).

As used herein, the term “small RNA” refers to several classes ofnon-coding ribonucleic acid (ncRNA). The term small RNA describes theshort chains of ncRNA produced in bacterial cells, animals, plants, andfungi. These short chains of ncRNA may be produced naturally within thecell or may be produced by the introduction of an exogenous sequencethat expresses the short chain or ncRNA. The small RNA sequences do notdirectly code for a protein, and differ in function from other RNA inthat small RNA sequences are only transcribed and not translated. Thesmall RNA sequences are involved in other cellular functions, includinggene expression and modification. Small RNA molecules are usually madeup of about 20 to 30 nucleotides. The small RNA sequences may be derivedfrom longer precursors. The precursors form structures that fold back oneach other in self-complementary regions; they are then processed by thenuclease Dicer in animals, DCL1 in plants, or other enzymes that processthe small RNA molecule.

Many types of small RNA exist either naturally or produced artificially,including microRNAs (miRNAs), short interfering RNAs (siRNAs), antisenseRNA, short hairpin RNA (shRNA), and small nucleolar RNAs (snoRNAs).Certain types of small RNA, such as microRNA and siRNA, are important ingene silencing and RNA interference (RNAi). Gene silencing is a processof genetic regulation in which a gene that would normally be expressedis “turned off” by an intracellular element, in this case, the smallRNA. The protein that would normally be formed by this geneticinformation is not formed due to interference, and the information codedin the gene is blocked from expression.

As used herein, the term “small RNA” encompasses RNA molecules describedin the literature as “tiny RNA” (Storz, (2002) Science 296:1260-3;Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic “small RNA”(sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic“noncoding RNA (ncRNA)”; “micro-RNA (miRNA)”; “small non-mRNA (snmRNA)”;“functional RNA (fRNA)”; “transfer RNA (tRNA)”; “catalytic RNA” [e.g.,ribozymes, including self-acylating ribozymes (Illangaskare et al.,(1999) RNA 5:1482-1489); “small nucleolar RNAs (snoRNAs)”; “tmRNA”(a.k.a. “10S RNA”, Muto et al., (1998) Trends Biochem Sci. 23:25-29; andGillet et al., (2001) Mol Microbiol. 42:879-885); RNAi moleculesincluding without limitation “small interfering RNA (siRNA)”,“endoribonuclease-prepared siRNA (e-siRNA)”, “short hairpin RNA(shRNA)”, and “small temporally regulated RNA (stRNA)”; “diced siRNA(d-siRNA)”, and aptamers, oligonucleotides and other synthetic nucleicacids that comprise at least one uracil base.

As used herein, the term “intron” is defined as any nucleic acidsequence comprised in a gene (or expressed nucleotide sequence ofinterest) that is transcribed but not translated. Introns includeuntranslated nucleic acid sequence within an expressed sequence of DNA,as well as corresponding sequence in RNA molecules transcribedtherefrom. A construct described herein can also contain sequences thatenhance translation and/or mRNA stability such as introns. An example ofone such intron is the first intron of gene II of the histone H3 variantof Arabidopsis thaliana or any other commonly known intron sequence.Introns can be used in combination with a promoter sequence to enhancetranslation and/or mRNA stability.

As used herein, the terms “5′ untranslated region” or “5′-UTR” isdefined as the untranslated segment in the 5′ terminus of pre-mRNAs ormature mRNAs. For example, on mature mRNAs, a 5′-UTR typically harborson its 5′ end a 7-methylguanosine cap and is involved in many processessuch as splicing, polyadenylation, mRNA export towards the cytoplasm,identification of the 5′ end of the mRNA by the translational machinery,and protection of the mRNAs against degradation.

As used herein, the terms “transcription terminator” is defined as thetranscribed segment in the 3′ terminus of pre-mRNAs or mature mRNAs. Forexample, longer stretches of DNA beyond “polyadenylation signal” site istranscribed as a pre-mRNA. This DNA sequence usually contains one ormore transcription termination signals for the proper processing of thepre-mRNA into mature mRNA.

As used herein, the term “3′ untranslated region” or “3′-UTR” is definedas the untranslated segment in a 3′ terminus of the pre-mRNAs or maturemRNAs. For example, on mature mRNAs this region harbors the poly-(A)tail and is known to have many roles in mRNA stability, translationinitiation, and mRNA export.

As used herein, the term “polyadenylation signal” designates a nucleicacid sequence present in mRNA transcripts that allows for transcripts,when in the presence of a poly-(A) polymerase, to be polyadenylated onthe polyadenylation site, for example, located 10 to 30 bases downstreamof the poly-(A) signal. Many polyadenylation signals are known in theart and are useful for the present invention. An exemplary sequenceincludes AAUAAA and variants thereof, as described in Loke J., et al.,(2005) Plant Physiology 138(3); 1457-1468.

The term “isolated” as used herein means having been removed from itsnatural environment, or removed from other compounds present when thecompound is first formed. The term “isolated” embraces materialsisolated from natural sources as well as materials (e.g., nucleic acidsand proteins) recovered after preparation by recombinant expression in ahost cell, or chemically-synthesized compounds such as nucleic acidmolecules, proteins, and peptides.

The term “purified”, as used herein relates to a molecule or compound ina form that is substantially free of contaminants normally associatedwith the molecule or compound in a native or natural environment, orsubstantially enriched in concentration relative to other compoundspresent when the compound is first formed, and means having beenincreased in purity as a result of being separated from other componentsof the original composition. The term “purified nucleic acid” is usedherein to describe a nucleic acid sequence which has been separated,produced apart from, or purified away from other biological compoundsincluding, but not limited to polypeptides, lipids and carbohydrates,while effecting a chemical or functional change in the component (e.g.,a nucleic acid may be purified from a chromosome by removing proteincontaminants and breaking chemical bonds connecting the nucleic acid tothe remaining DNA in the chromosome).

As used herein, the terms “Homology-Based Gene Silencing” or “HBGS” aregeneric terms that include both transcriptional gene silencing andposttranscriptional gene silencing. Silencing of a target locus by anunlinked silencing locus can result from transcription inhibition(Transcriptional Gene Silencing; TGS) or mRNA degradation(Post-Transcriptional Gene Silencing; PTGS), owing to the production ofdouble-stranded RNA (dsRNA) corresponding to promoter or transcribedsequences, respectively. Involvement of distinct cellular components ineach process suggests that dsRNA-induced TGS and PTGS likely resultsfrom the diversification of an ancient common mechanism. However, astrict comparison of TGS and PTGS has been difficult to achieve becauseit generally relies on the analysis of distinct silencing loci. A singletransgene locus can be described to trigger both TGS and PTGS, owing tothe production of dsRNA corresponding to promoter and transcribedsequences of different target genes.

As used herein, the terms “nucleic acid molecule”, “nucleic acid”, or“polynucleotide” (all three terms are synonymous with one another) referto a polymeric form of nucleotides, which may include both sense andanti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms, andmixed polymers thereof. “A nucleotide” may refer to a ribonucleotide,deoxyribonucleotide, or a modified form of either type of nucleotide. Anucleic acid molecule is usually at least 10 bases in length, unlessotherwise specified. The terms may refer to a molecule of RNA or DNA ofindeterminate length. The terms include single- and double-strandedforms of DNA. A nucleic acid molecule may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Transcription proceeds in a 5′ to 3′ manner along a DNA strand. Thismeans that RNA is made by sequential addition ofribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain(with a requisite elimination of the pyrophosphate). In either a linearor circular nucleic acid molecule, discrete elements (e.g., particularnucleotide sequences) may be referred to as being “upstream” relative toa further element if they are bonded or would be bonded to the samenucleic acid in the 5′ direction from that element. Similarly, discreteelements may be “downstream” relative to a further element if they areor would be bonded to the same nucleic acid in the 3′ direction fromthat element.

As used herein, the term “base position”, refers to the location of agiven base or nucleotide residue within a designated nucleic acid. Adesignated nucleic acid may be defined by alignment with a referencenucleic acid.

As used herein, the term “hybridization” refers to a process whereoligonucleotides and their analogs hybridize by hydrogen bonding, whichincludes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary bases. Generally, nucleic acid molecules consistof nitrogenous bases that are either pyrimidines (cytosine (C), uracil(U), and thymine (T)) or purines (adenine (A) and guanine (G)). Thesenitrogenous bases form hydrogen bonds between a pyrimidine and a purine,and bonding of a pyrimidine to a purine is referred to as “basepairing.” More specifically, A will hydrogen bond to T or U, and G willbond to C. “Complementary” refers to the base pairing that occursbetween two distinct nucleic acid sequences or two distinct regions ofthe same nucleic acid sequence.

As used herein, the terms “specifically hybridizable” and “specificallycomplementary” refers to a sufficient degree of complementarity suchthat stable and specific binding occurs between an oligonucleotide andthe DNA or RNA target. Oligonucleotides need not be 100% complementaryto its target sequence to specifically hybridize. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of an oligonucleotide to non-target sequencesunder conditions where specific binding is desired, for example underphysiological conditions in the case of in vivo assays or systems. Suchbinding is referred to as specific hybridization. Hybridizationconditions resulting in particular degrees of stringency will varydepending upon the nature of the chosen hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength(especially Na⁺ and/or Mg²⁺ concentration) of a hybridization bufferwill contribute to the stringency of hybridization, though wash timesalso influence stringency. Calculations regarding hybridizationconditions required for attaining particular degrees of stringency arediscussed in Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, chs. 9 and 11.

As used herein, the term “stringent conditions” encompasses conditionsunder which hybridization will only occur if there is less than 50%mismatch between the hybridization molecule and the DNA target.“Stringent conditions” include further particular levels of stringency.Thus, as used herein, “moderate stringency” conditions are those underwhich molecules with more than 50% sequence mismatch will not hybridize;conditions of “high stringency” are those under which sequences withmore than 20% mismatch will not hybridize; and conditions of “very highstringency” are those under which sequences with more than 10% mismatchwill not hybridize. In particular embodiments, stringent conditions caninclude hybridization at 65° C., followed by washes at 65° C. with0.1×SSC/0.1% SDS for 40 minutes. The following are representative,non-limiting hybridization conditions:

-   -   Very High Stringency: hybridization in 5×SSC buffer at 65° C.        for 16 hours; wash twice in 2×SSC buffer at room temperature for        15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for        20 minutes each.    -   High Stringency: Hybridization in 5-6×SSC buffer at 65-70° C.        for 16-20 hours; wash twice in 2×SSC buffer at room temperature        for 5-20 minutes each; and wash twice in 1×SSC buffer at        55-70° C. for 30 minutes each.    -   Moderate Stringency: Hybridization in 6×SSC buffer at room        temperature to 55° C. for 16-20 hours; wash at least twice in        2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes        each.

In an embodiment, specifically hybridizable nucleic acid molecules canremain bound under very high stringency hybridization conditions. In anembodiment, specifically hybridizable nucleic acid molecules can remainbound under high stringency hybridization conditions. In an embodiment,specifically hybridizable nucleic acid molecules can remain bound undermoderate stringency hybridization conditions.

As used herein, the term “oligonucleotide” refers to a short nucleicacid polymer. Oligonucleotides may be formed by cleavage of longernucleic acid segments, or by polymerizing individual nucleotideprecursors. Automated synthesizers allow the synthesis ofoligonucleotides up to several hundred base pairs in length. Becauseoligonucleotides may bind to a complementary nucleotide sequence, theymay be used as probes for detecting DNA or RNA. Oligonucleotidescomposed of DNA (oligodeoxyribonucleotides) may be used in PCR, atechnique for the amplification of small DNA sequences. In PCR, anoligonucleotide is typically referred to as a “primer”, which allows aDNA polymerase to extend the oligonucleotide and replicate thecomplementary strand.

As used herein, the terms “Polymerase Chain Reaction” or “PCR” refer toa procedure or technique in which minute amounts of nucleic acid, RNAand/or DNA, are amplified as described in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond needs to be available, such that oligonucleotide primers canbe designed; these primers will be identical or similar in sequence toopposite strands of the template to be amplified. The 5′ terminalnucleotides of the two primers may coincide with the ends of theamplified material. PCR can be used to amplify specific RNA sequences,specific DNA sequences from total genomic DNA, and cDNA transcribed fromtotal cellular RNA, bacteriophage or plasmid sequences, etc. Seegenerally Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263(1987); Erlich, ed., PCR Technology, (Stockton Press, NY, 1989).

As used herein, the term “primer” refers to an oligonucleotide capableof acting as a point of initiation of synthesis along a complementarystrand when conditions are suitable for synthesis of a primer extensionproduct. The synthesizing conditions include the presence of fourdifferent deoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer, which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

As used herein, the term “probe” refers to an oligonucleotide thathybridizes to a target sequence. In the TaqMan® or TaqMan®-style assayprocedure, the probe hybridizes to a portion of the target situatedbetween the annealing site of the two primers. A probe includes abouteight nucleotides, about ten nucleotides, about fifteen nucleotides,about twenty nucleotides, about thirty nucleotides, about fortynucleotides, or about fifty nucleotides. In some embodiments, a probeincludes from about eight nucleotides to about fifteen nucleotides. Aprobe can further include a detectable label, e.g., a fluorophore(Texas-Red®, Fluorescein isothiocyanate, etc.,). The detectable labelcan be covalently attached directly to the probe oligonucleotide, e.g.,located at the probe's 5′ end or at the probe's 3′ end. A probeincluding a fluorophore may also further include a quencher, e.g., BlackHole Quencher™, Iowa Black™, etc.

As used herein, the terms “sequence identity” or “identity” can be usedinterchangeably and refer to nucleic acid residues or amino acidsequences in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” refers to avalue determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences or amino acid sequences) over a comparisonwindow, wherein the portion of a sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to a referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. A percentage is calculated bydetermining the number of positions at which an identical nucleic acidor amino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity. Methods foraligning sequences for comparison are well known. Various programs andalignment algorithms are described in, for example: Smith and Waterman(1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol.48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444;Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang etal. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) MethodsMol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett.174:247-50.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990) J. Mol. Biol.215:403-10) is available from several sources, including the NationalCenter for Biotechnology Information (Bethesda, Md.), and on theinternet, for use in connection with several sequence analysis programs.A description of how to determine sequence identity using this programis available on the internet under the “help” section for BLAST™. Forcomparisons of nucleic acid sequences, the “Blast 2 sequences” functionof the BLAST™ (Blastn) program may be employed using the defaultparameters. Nucleic acid sequences with even greater similarity to thereference sequences will show increasing percentage identity whenassessed by this method.

As used herein, the term “operably linked” refers to two components thathave been placed into a functional relationship with one another. Theterm, “operably linked”, when used in reference to a regulatory sequenceand a coding sequence, means that the regulatory sequence affects theexpression of the linked coding sequence. “Regulatory sequences”,“regulatory elements”, or “control elements”, refer to nucleic acidsequences that influence the timing and level/amount of transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include promoters; translation leadersequences; 5′ and 3′ untranslated regions, introns; enhancers; stem-loopstructures; repressor binding sequences; termination sequences;polyadenylation recognition sequences; etc. Particular regulatorysequences may be located upstream and/or downstream of a coding sequenceoperably linked thereto. Also, particular regulatory sequences operablylinked to a coding sequence may be located on the associatedcomplementary strand of a double-stranded nucleic acid molecule. Linkingcan be accomplished by ligation at convenient restriction sites. If suchsites do not exist, synthetic oligonucleotide adaptors or linkers areused in accordance with conventional practice. However, elements neednot be contiguous to be operably linked.

As used herein, the term “transformation” encompasses all techniques bywhich a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation;lipofection; microinjection (Mueller et al. (1978) Cell 15:579-85);Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS®-mediatedtransformation; and microprojectile bombardment. Transformation may bestable, wherein the nucleic acid molecule is integrated within thegenome of the plant, and is subsequently passed from generation togeneration. Comparatively, transformation may be transient, wherein thenucleic acid molecule is localized within the cytoplasm or nucleus ofthe cell and is not integrated within the genome of the plant. Such atransient transformant may result in the expression of protein or a geneproduct from coding sequences present on the nucleic acid molecule.

As used herein, the term “transduce” refers to a process where a virustransfers nucleic acid into a cell.

The terms “polylinker” or “multiple cloning site” as used herein definesa cluster of one or more Type −2 restriction enzyme sites. Adjacentrestriction sites may be included in a polylinker and are typicallylocated within 10 nucleotides of one another on a nucleic acid sequence.Constructs comprising a polylinker are utilized for the insertion and/orexcision of nucleic acid sequences such as the coding region of a gene,a ribosomal binding sequence, an intron, or a 5′-UTR.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence. Type −2 restrictionenzymes recognize and cleave DNA at the same site, and include but arenot limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstIand SmaI.

The term “vector” is used interchangeably with the terms “construct”,“cloning vector” and “expression vector” and means the vehicle by whicha DNA or RNA sequence (e.g. a foreign gene) can be introduced into ahost cell, so as to transform the host and promote expression (e.g.transcription and translation) of the introduced sequence. A “non-viralvector” is intended to mean any vector that does not comprise a virus orretrovirus. In some embodiments a “vector” is a sequence of DNAcomprising at least one origin of DNA replication and at least oneselectable marker gene. Examples include, but are not limited to, aplasmid, cosmid, bacteriophage, bacterial artificial chromosome (BAC),or virus that carries exogenous DNA into a cell. A vector can alsoinclude one or more genes, antisense molecules, and/or selectable markergenes and other genetic elements known in the art. A vector maytransduce, transform, or infect a cell, thereby causing the cell toexpress the nucleic acid molecules and/or proteins encoded by thevector. The term “plasmid” defines a circular strand of nucleic acidcapable of autosomal replication in either a prokaryotic or a eukaryotichost cell. The term includes nucleic acid which may be either DNA or RNAand may be single- or double-stranded. The plasmid of the definition mayalso include the sequences which correspond to a bacterial origin ofreplication.

The term “selectable marker gene” as used herein defines a gene or otherexpression cassette which encodes a protein which facilitatesidentification of cells into which the selectable marker gene isinserted. For example a “selectable marker gene” encompasses reportergenes as well as genes used in plant transformation to, for example,protect plant cells from a selective agent or provideresistance/tolerance to a selective agent. In one embodiment only thosecells or plants that receive a functional selectable marker are capableof dividing or growing under conditions having a selective agent.Examples of selective agents can include, for example, antibiotics,including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin,and hygromycin. These selectable markers include neomycinphosphotransferase (npt II), which expresses an enzyme conferringresistance to the antibiotic kanamycin, and genes for the relatedantibiotics neomycin, paromomycin, gentamicin, and G418, or the gene forhygromycin phosphotransferase (hpt), which expresses an enzymeconferring resistance to hygromycin. Other selectable marker genes caninclude genes encoding herbicide resistance including bar or pat(resistance against glufosinate ammonium or phosphinothricin),acetolactate synthase (ALS, resistance against inhibitors such assulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs),pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyltriazolinones that prevent the first step in the synthesis of thebranched-chain amino acids), glyphosate, 2,4-D, and metal resistance orsensitivity. Examples of “reporter genes” that can be used as aselectable marker gene include the visual observation of expressedreporter gene proteins such as proteins encoding β-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT),alkaline phosphatase, and the like. The phrase “marker-positive” refersto plants that have been transformed to include a selectable markergene.

As used herein, the term “detectable marker” refers to a label capableof detection, such as, for example, a radioisotope, fluorescentcompound, bioluminescent compound, a chemiluminescent compound, metalchelator, or enzyme. Examples of detectable markers include, but are notlimited to, the following: fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In anembodiment, a detectable marker can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, the term “detecting” is used in the broadest sense toinclude both qualitative and quantitative measurements of a specificmolecule, for example, measurements of a specific polypeptide.

As used herein, the terms “cassette”, “expression cassette” and “geneexpression cassette” refer to a segment of DNA that can be inserted intoa nucleic acid or polynucleotide at specific restriction sites or byhomologous recombination. A segment of DNA comprises a polynucleotidethat encodes a polypeptide of interest, and the cassette and restrictionsites are designed to ensure insertion of the cassette in the properreading frame for transcription and translation. In an embodiment, anexpression cassette can include a polynucleotide that encodes apolypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.In an embodiment, a gene expression cassette may also include elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein a “linker” or “spacer” is a bond, molecule or group ofmolecules that binds two separate entities to one another. Linkers andspacers may provide for optimal spacing of the two entities or mayfurther supply a labile linkage that allows the two entities to beseparated from each other. Labile linkages include photocleavablegroups, acid-labile moieties, base-labile moieties and enzyme-cleavablegroups.

As used herein, the term “control” refers to a sample used in ananalytical procedure for comparison purposes. A control can be“positive” or “negative”. For example, where the purpose of ananalytical procedure is to detect a differentially expressed transcriptor polypeptide in cells or tissue, it is generally preferable to includea positive control, such as a sample from a known plant exhibiting thedesired expression, and a negative control, such as a sample from aknown plant lacking the desired expression.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. A class of plant that canbe used in the present invention is generally as broad as the class ofhigher and lower plants amenable to mutagenesis including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns andmulticellular algae. Thus, “plant” includes dicot and monocot plants.The term “plant parts” include any part(s) of a plant, including, forexample and without limitation: seed (including mature seed and immatureseed); a plant cutting; a plant cell; a plant cell culture; a plantorgan (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots,stems, and explants). A plant tissue or plant organ may be a seed,protoplast, callus, or any other group of plant cells that is organizedinto a structural or functional unit. A plant cell or tissue culture maybe capable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. In contrast, some plant cells are not capable ofbeing regenerated to produce plants. Regenerable cells in a plant cellor tissue culture may be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant,comprising a protoplast and a cell wall. A plant cell may be in the formof an isolated single cell, or an aggregate of cells (e.g., a friablecallus and a cultured cell), and may be part of a higher organized unit(e.g., a plant tissue, plant organ, and plant). Thus, a plant cell maybe a protoplast, a gamete producing cell, or a cell or collection ofcells that can regenerate into a whole plant. As such, a seed, whichcomprises multiple plant cells and is capable of regenerating into awhole plant, is considered a “plant cell” in embodiments herein.

The term “protoplast”, as used herein, refers to a plant cell that hadits cell wall completely or partially removed, with the lipid bilayermembrane thereof naked, and thus includes protoplasts, which have theircell wall entirely removed, and spheroplasts, which have their cell wallonly partially removed, but is not limited thereto. Typically, aprotoplast is an isolated plant cell without cell walls which has thepotency for regeneration into cell culture or a whole plant.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample: Lewin, Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers(ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

EMBODIMENTS

As disclosed herein novel recombinant constructs are provided forexpressing a non-chlorophyll a/b transgene using the regulatorysequences of a chlorophyll a/b gene from Zea mays. These constructs canbe used to transform cells, including plant cells, to produce completeorganisms that express the transgene gene product in their cells.

Plant promoters used for basic research or biotechnological applicationare generally unidirectional, directing only one gene that has beenfused at its 3′ end (downstream). It is often necessary to introducemultiple genes into plants for metabolic engineering and trait stackingand therefore, multiple promoters are typically required in transgeniccrops to drive the expression of multiple genes.

Development of transgenic products is becoming increasingly complex,which requires stacking multiple transgenes into a single locus.Traditionally, each transgene usually requires a promoter for expressionwherein multiple promoters are required to express different transgeneswithin one gene stack. This frequently leads to repetitive use of thesame promoter within one transgene stack to obtain similar levels ofexpression patterns of different transgenes for expression of a singlepolygenic trait. Multi-gene constructs driven by the same promoter areknown to cause gene silencing resulting in less efficacious transgenicproducts in the field. Excess of transcription factor (TF)-binding sitesdue to promoter repetition can cause depletion of endogenous TFs leadingto transcriptional inactivation. The silencing of transgenes will likelyundesirably affect performance of a transgenic plant produced to expresstransgenes. Repetitive sequences within a transgene may lead to geneintra locus homologous recombination resulting in polynucleotiderearrangements.

Moreover, tissue specific (i.e., tissue-preferred) or organ specificpromoters drive gene expression in a certain tissue such as in thekernel, root, leaf or tapetum of the plant. Tissue and developmentalstage specific promoters derive the expression of genes, which areexpressed in particular tissues or at particular time periods duringplant development. Tissue specific promoters are required for certainapplications in the transgenic plants industry and are desirable as theypermit specific expression of heterologous genes in a tissue and/ordevelopmental stage selective manner, indicating expression of theheterologous gene differentially at a various organs, tissues and/ortimes, but not in other. For example, increased resistance of a plant toinfection by soil-borne pathogens might be accomplished by transformingthe plant genome with a pathogen-resistance gene such thatpathogen-resistance protein is robustly expressed within the roots ofthe plant. Alternatively, it may be desirable to express a transgene inplant tissues that are in a particular growth or developmental phasesuch as, for example, cell division or elongation. Another applicationis the desirability of using tissue specific promoters, e.g. such thatwould confine the expression of the transgenes encoding an agronomictrait in developing xylem. One particular problem remaining in theidentification of tissue specific promoters is how to identify thepotentially most important genes and their corresponding promoters, andto relate these to specific developmental properties of the cell.Another problem is to clone all relevant cis-acting transcriptionalcontrol elements so that the cloned DNA fragment drives transcription inthe wanted specific expression pattern. A particular problem is toidentify tissue-specific promoters, related to specific cell types,developmental stages and/or functions in the plant that are notexpressed in other plant tissues.

It is desirable to use diversified promoters for the expression ofdifferent transgenes in a gene stack. In an embodiment, regulatoryelements obtained from the Zea mays plant species can drivetranscription of multiple transcription units, including transgenes,RNAi, artificial miRNA, or hairpin-loop RNA sequences. As a furtherembodiment, a chlorophyll a/b promoter can be obtained from Zea mays todrive transcription of multiple transcription units, including atransgene, RNAi, artificial miRNA, or hairpin-loop RNA sequences. In yetanother embodiment, a chlorophyll a/b promoter can be obtained from Zeamays to drive transcription of multiple transcription units, including atransgene, RNAi, artificial miRNA, or hairpin-loop RNA sequences inleaf, cob, silk, kernel, stem, husk and pollen tissues of a plant.

Provided are methods and constructs using gene regulatory elementsisolated from Zea mays to express transgenes in plant. In an embodiment,a Zea mays promoter can be an isolated promoter of SEQ ID NO:1.

In an embodiment, a nucleic acid vector (i.e., construct) is providedcomprising a promoter. In an embodiment, a promoter can be a Zea maysgene promoter. In an embodiment, a nucleic acid vector is providedcomprising a promoter, wherein the promoter is at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:1. In an embodiment, a nucleic acid vectorcomprises a Zea mays gene promoter that is operably linked to apolylinker. In an embodiment, a gene expression cassette is providedcomprising a Zea mays promoter that is operably linked to anon-chlorophyll a/b transgene. In one embodiment the promoter consistsof SEQ ID NO: 1. In an illustrative embodiment, a gene expressioncassette comprises a Zea mays promoter that is operably linked to atransgene, wherein the transgene can be an insecticidal resistancetransgene, an herbicide tolerance transgene, a nitrogen use efficiencytransgene, a water use efficiency transgene, a nutritional qualitytransgene, a DNA binding transgene, a selectable marker transgene, orcombinations thereof.

In addition to a promoter, a 3′-untranslated gene region (i.e., 3′UTR)or terminator is needed for transcription termination andpolyadenylation of the mRNA. Proper transcription termination andpolyadenylation of mRNA is important for stable expression of transgene.The transcription termination becomes more critical for multigene stacksto avoid transcription read-through into next transgene. Similarly,non-polyadenylated aberrant RNA (aRNA) is a substrate for plantRNA-dependent RNA polymerases (RdRPs) to convert aRNA into doublestranded RNA (dsRNA) leading to small RNA production and transgenesilencing. Strong transcription terminators therefore are very usefulboth for single gene and multiple gene stacks. While a promoter isnecessary to drive transcription, a 3′-UTR gene region can terminatetranscription and initiate polyadenylation of a resulting mRNAtranscript for translation and protein synthesis. A 3′-UTR gene regionaids stable expression of a transgene.

In accordance with one embodiment a nucleic acid construct is providedcomprising a Zea mays transcription terminator. In an embodiment, theZea mays transcription terminator is provided comprising a transcriptionterminator, wherein the transcription terminator is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to SEQ ID NO:6. In an embodiment, a nucleic acid construct isprovided comprising a Zea mays transcription terminator that is operablylinked to a polylinker. In an embodiment, a gene expression cassette isprovided comprising a Zea mays transcription terminator that is operablylinked to the 3′ end of a non-chlorophyll a/b transgene. In oneembodiment the transcription terminator consists of SEQ ID NO:6. In anillustrative embodiment, a gene expression cassette comprises a Zea maystranscription terminator that is operably linked to a transgene, whereinthe transgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. Inone embodiment a nucleic acid vector is provided comprising atranscription terminator operably linked to either a polylinkersequence, a non-chlorophyll a/b transgene or a combination of both,wherein the transcription terminator comprises SEQ ID NO:6 or a sequencethat has 90% sequence identity with SEQ ID NO:6. In one embodiment thetranscription terminator is less than 1 kb in length, and in a furtherembodiment the transcription terminator consists of the 3′UTR sequenceof SEQ ID NO:6.

In an embodiment, a nucleic acid construct is provided comprising a Zeamays promoter as described herein and a 3′-UTR. In an embodiment, thenucleic acid construct comprises a Zea mays 3′-UTR. In an embodiment,the Zea mays 3′-UTR is a Zea mays 3′-UTR. In an embodiment, a 3′-UTR canbe the Zea mays 3′-UTR of SEQ ID NO:6.

In an embodiment, a nucleic acid construct is provided comprising a Zeamays promoter as described herein and a 3′-UTR, wherein the 3′-UTR is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:6. In an embodiment, a nucleicacid construct is provided comprising a Zea mays promoter as describedherein and the 3′-UTR wherein the Zea mays promoter and 3′-UTR are bothoperably linked to opposite ends of a polylinker. In an embodiment, agene expression cassette is provided comprising a Zea mays promoter asdescribed herein and a 3′-UTR, wherein the Zea mays promoter and 3′-UTRare both operably linked to opposite ends of a non-chlorophyll a/btransgene. In one embodiment the a 3′-UTR, consists of SEQ ID NO:6. Inone embodiment, a gene expression cassette is provided comprising a Zeamays promoter as described herein and a 3′-UTR, wherein the Zea mayspromoter comprises SEQ ID NO:1 and the 3′-UTR comprises SEQ ID NO:6wherein the promoter and 3′-UTR are operably linked to opposite ends ofa non-cholorophyll a/b transgene. In one embodiment the a 3′-UTR,consists of SEQ ID NO:6. In one embodiment the promoter consists of SEQID NO:1 or SEQ ID NO:5 and the 3′-UTR, consists of SEQ ID NO:6. In anillustrative embodiment, a gene expression cassette comprises a Zea mays3′-UTR that is operably linked to a transgene, wherein the transgene canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof. In a furtherembodiment the transgene is operably linked to a Zea mays promoter and a3′-UTR from the same gene isolated from Zea mays.

In one embodiment a vector is provided comprising a first transgeneand/or polylinker and a second transgene and/or polylinker wherein thefirst transgene and/or polylinker is operably linked to a promotercomprising a sequence selected from the group consisting of SEQ ID NO:1and SEQ ID NO:5 and operably linked to a 3′-UTR, comprising a sequenceselected from the group consisting of SEQ ID NO:6.

Transgene expression may also be regulated by an intron region locateddownstream of the promoter sequence. Both a promoter and an intron canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of an intron can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. Anintron gene region aids stable expression of a transgene.

In an embodiment, a nucleic acid construct is provided comprising a Zeamays promoter as described herein and an intron. In one embodiment theintron is operably linked to the 3′ end of the promoter. In anembodiment, a nucleic acid construct is provided comprising a Zea maysintron operably linked to the 3′ end of a Zea mays promoter or aderivative of such promoter sequence. In an embodiment, the Zea maysintron is a Zea mays chlorophyll a/b intron, or a derivative of suchintron sequence.

In an embodiment, an intron can be the Zea mays chlorophyll a/b intronof SEQ ID NO:2. In another embodiment, an intron can be the Zea mayschlorophyll a/b intron of SEQ ID NO:3. In an embodiment, a nucleic acidconstruct is provided comprising a Zea mays promoter as described hereinand an intron, wherein the intron is at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical toSEQ ID NO:2 or SEQ ID NO:3. In an embodiment, a nucleic acid constructis provided comprising a Zea mays promoter as described herein, anintron sequence and a polylinker wherein the promoter and intron areoperably linked to a polylinker. In an embodiment, a gene expressioncassette is provided comprising a Zea mays promoter as described herein,an intron sequence and a non-chlorophyll a/b transgene wherein thepromoter and intron are operably linked to the 5′ end of the transgene.Optionally the construct further comprises a 3′-UTR that is operablylinked to the 3′ end of the non-chlorophyll a/b transgene or polylinker.In one embodiment the promoter and 3′-UTR sequences are selected fromthose described herein and the intron sequence consists of SEQ ID NO:2or SEQ ID NO:3. In an embodiment, a gene expression cassette comprises aZea mays intron that is operably linked to a promoter, wherein thepromoter is a Zea mays promoter of SEQ ID NO:1, or a promoter thatoriginates from a plant (e.g., Zea mays ubiquitin 1 promoter), a virus(e.g., Cassava vein mosaic virus promoter) or a bacteria (e.g.,Agrobacterium tumefaciens delta mas). In an illustrative embodiment, agene expression cassette comprises a Zea mays intron that is operablylinked to a transgene, wherein the transgene can be an insecticidalresistance transgene, an herbicide tolerance transgene, a nitrogen useefficiency transgene, a water use efficiency transgene, a nutritionalquality transgene, a DNA binding transgene, a selectable markertransgene, or combinations thereof.

Transgene expression may also be regulated by a 5′-UTR region locateddownstream of the promoter sequence. Both a promoter and a 5′-UTR canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of a 5′-UTR can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. A5′-UTR gene region aids stable expression of a transgene.

In an embodiment, a nucleic acid construct is provided comprising a Zeamays promoter as described herein and a 5′-UTR. In one embodiment the5′-UTR is operably linked to the 3′ end of the promoter. In anembodiment, a nucleic acid construct is provided comprising a Zea mays5′-UTR operably linked to the 3′ end of a chlorophyll a/b promoterisolated from Panicum Zea mays or a derivative of such promotersequence. In a further embodiment the 3′ end of the 5′-UTR is operablylinked to the 5′ end of a Zea mays intron, as described herein.

In an embodiment, a 5′-UTR can be the Zea mays 5′-UTR of SEQ ID NO:4. Inan embodiment, a nucleic acid construct is provided comprising a Zeamays promoter as disclosed herein and a 5′-UTR, wherein the 5′-UTR is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical SEQ ID NO:4. In an embodiment, a nucleic acidconstruct is provided comprising Zea mays promoter, wherein the promoteris at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identical to SEQ ID NO:4, and a 5′-UTR operablylinked to a polylinker. In an embodiment, a gene expression cassette isprovided comprising a Zea mays promoter, wherein the promoter is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:4, and a 5′-UTR sequences operablylinked to a non-chlorophyll a/b transgene. Optionally, the construct canfurther comprise a Zea mays intron as disclosed herein operably linkedto the 3′ end of the 5′-UTR and the 5′ end of the non-chlorophyll a/btransgene and optionally further comprising a 3′-UTR that is operablylinked to the 3′ end of the non-chlorophyll a/b transgene. In oneembodiment the promoter, intron and 3′-UTR sequences are selected fromthose described herein and the 5′-UTR sequence consists of SEQ ID NO:4.In one embodiment the 3′-UTR consists of SEQ ID NO:6.

In an embodiment, a gene expression cassette comprises a Zea mays 5′-UTRthat is operably linked to a promoter, wherein the promoter is a Zeamays promoter of SEQ ID NO:1, or a promoter that originates from a plant(e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava veinmosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciensdelta mas). In an illustrative embodiment, a gene expression cassettecomprises a Zea mays 5′-UTR that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In one embodiment a nucleic acid construct is provided comprising apromoter and a polylinker and optionally one or more of the followingelements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ untranslated region,

wherein

the promoter consists of SEQ ID NO:1 or a sequence having 98% sequenceidentity with SEQ ID NO:1;

the 5′ untranslated region consists of SEQ ID NO:4 or a sequence having98% sequence identity with SEQ ID NO:4;

the intron consists of SEQ ID NO:2, or SEQ ID NO:3 or a sequence having98% sequence identity with SEQ ID NO:2, or SEQ ID NO:3;

the 3′ untranslated region consists of SEQ ID NO:6 or a sequence having98% sequence identity with SEQ ID NO:6;

further wherein said promoter is operably linked to said polylinker andeach optional element, when present, is also operably linked to both thepromoter and the polylinker.

In one embodiment a nucleic acid construct is provided comprising apromoter and a non-chlorophyll a/b transgene and optionally one or moreof the following elements:

a) a 5′ untranslated region;

b) an intron; and

c) a 3′ untranslated region,

wherein

the promoter consists of SEQ ID NO:1 or a sequence having 98% sequenceidentity with SEQ ID NO:1;

the 5′ untranslated region consists of SEQ ID NO:4 or a sequence having98% sequence identity with SEQ ID NO:4;

the intron consists of SEQ ID NO:2, or SEQ ID NO:3 or a sequence having98% sequence identity with SEQ ID NO:2, or SEQ ID NO:3;

the 3′ untranslated region consists of SEQ ID NO:6, or a sequence having98% sequence identity with SEQ ID NO:6;

further wherein said promoter is operably linked to said transgene andeach optional element, when present, is also operably linked to both thepromoter and the transgene. In a further embodiment a transgenic cell isprovided comprising the nucleic acid construct disclosed immediatelyabove. In one embodiment the transgenic cell is a plant cell, and in afurther embodiment a plant is provided wherein the plant comprises saidtransgenic cells.

In accordance with one embodiment transgene expression is regulated by apromoter operably linked to an intron and 5′-UTR region, wherein theintron and 5′-UTR region are located downstream of the promotersequence. A promoter operably linked to an intron and 5′-UTR region canbe used to drive transgene expression. While a promoter is necessary todrive transcription, the presence of the intron and 5′-UTR can increaseexpression levels resulting in mRNA transcript for translation andprotein synthesis.

In an embodiment, a gene expression cassette comprises a promoteroperably linked to a 5′-UTR and intron region. In an embodiment, a geneexpression cassette comprises a Zea mays promoter operably linked to aZea mays 5′-UTR and Zea mays intron. In an embodiment, the Zea mayspromoter operably linked to a 5′-UTR and intron region is operablylinked to an intron and 5′-UTR.

In an embodiment, a promoter operably linked to a 5′-UTR and intron canbe the Zea mays promoter operably linked to an intron and 5′-UTR. In oneembodiment the promoter comprises or consists of the sequence of SEQ IDNO:5. In an embodiment, a nucleic acid construct is provided comprisinga promoter operably linked to an intron and 5′-UTR. In one embodimentthe construct comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:5. Inone embodiment, a nucleic acid construct is provided comprising a Zeamays promoter sequence comprising or consisting of a sequence at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:5 operably linked to a polylinker.Optionally, the construct can further comprise 3′-UTR that is operablylinked to the 3′ end of the polylinker. In an embodiment, a geneexpression cassette is provided comprising a Zea mays promoter sequencewherein the promoter sequence comprises or consists of a sequence atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:5 operably linked to anon-chlorophyll a/b transgene. Optionally, the construct can furthercomprise 3′-UTR that is operably linked to the 3′ end of thenon-chlorophyll a/b transgene. In one embodiment the 3′-UTR sequenceconsists of SEQ ID NO:6. In an illustrative embodiment, the transgenecan be an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof. In one embodimentthe transgene is an herbicide resistance gene. In one embodiment avector is provided comprising 1, 2, 3 or 4 promoter sequencesindependently selected from the group consisting of SEQ ID NO:1 or SEQID NO:5.

In an embodiment, a gene expression cassette comprises a Zea mayspromoter, a Zea mays 5′-UTR, a Zea mays intron, and a Zea mays 3′-UTR.In an embodiment, a gene expression cassette comprises: a) a promoter,wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1; b) a3′-UTR, wherein the 3′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ IDNO:6; c) a 5′-UTR, wherein the 5′-UTR is at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identicalto SEQ ID NO:4; or, d) an intron, wherein the intron is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or100% identical to SEQ ID NO:2, or SEQ ID NO:3.

For example, a gene expression cassette may include both a promoter, anintron, and a 5′-UTR wherein the promoter is a polynucleotide of SEQ IDNO:1, the intron is a polynucleotide of SEQ ID NO:2 or SEQ ID NO:3, andthe 5′-UTR is a polynucleotide of SEQ ID NO:4. Furthermore, a geneexpression cassette may include both a promoter, an intron, a 5′-UTR,and a 3′-UTR wherein the promoter is a polynucleotide of SEQ ID NO:1,the intron is a polynucleotide of SEQ ID NO:2 or SEQ ID NO:3, the 5′-UTRis a polynucleotide of SEQ ID NO:4, and the 3′-UTR is a polynucleotideof SEQ ID NO:6. In addition, a gene expression cassette may include botha promoter, and a 3′-UTR wherein the promoter is a polynucleotide of SEQID NO:1 and a 3′-UTR of SEQ ID NO:6

In an embodiment, a gene expression cassette comprises a Zea mayspromoter, Zea mays 5′-UTR, and a Zea mays 3′-UTR that are operablylinked to a non-chlorophyll a/b transgene.

A promoter, an intron, a 5′-UTR, and 3′-UTR can be operably linked todifferent transgenes within a gene expression cassette when a geneexpression cassette includes one or more transgenes. In an illustrativeembodiment, a gene expression cassette comprises a Zea mays promoterthat is operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In an illustrativeembodiment, a gene expression cassette comprises a Zea mays promoter, aZea mays intron, and a Zea mays 5′-UTR that are operably linked to atransgene, wherein the transgene can be an insecticidal resistancetransgene, an herbicide tolerance transgene, a nitrogen use efficiencytransgene, a water use efficiency transgene, a nutritional qualitytransgene, a DNA binding transgene, a selectable marker transgene, orcombinations thereof. In an illustrative embodiment, a gene expressioncassette comprises a Zea mays 3′-UTR that is operably linked to atransgene, wherein the transgene encodes for a gene product thatenhances insecticidal resistance, herbicide tolerance, nitrogen useefficiency, water use efficiency, nutritional quality or combinationsthereof.

A Zea mays intron and a 5′-UTR can be operably linked to differentpromoters within a gene expression cassette. In an illustrativeembodiment, the promoters originate from a plant (e.g., Zea maysubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic viruspromoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). Inan illustrative embodiment, a gene expression cassette comprises a Zeamays promoter that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof.

In an embodiment, a vector comprises a gene expression cassette asdisclosed herein. In an embodiment, a vector can be a plasmid, a cosmid,a bacterial artificial chromosome (BAC), a bacteriophage, a virus, or anexcised polynucleotide fragment for use in direct transformation or genetargeting such as a donor DNA.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene cassette wherein the recombinant genecassette comprises a Zea mays promoter of SEQ ID NO:1 operably linked toa polylinker sequence, a non-chlorophyll a/b transgene or combinationthereof. In one embodiment the recombinant gene cassette comprises a Zeamays promoter of SEQ ID NO:1 operably linked to a non-chlorophyll a/btransgene. In one embodiment the recombinant gene cassette comprises aZea mays promoter of SEQ ID NO:1 as disclosed herein operably linked toa polylinker sequence. The polylinker is operably linked to the a Zeamays promoter of SEQ ID NO:1 in a manner such that insertion of a codingsequence into one of the restriction sites of the polylinker willoperably link the coding sequence allowing for expression of the codingsequence when the vector is transfected into a host cell.

In accordance with one embodiment the Zea mays promoter comprises SEQ IDNO:1 or a sequence that has 90, 95 or 99% sequence identity with SEQ IDNO: 35. In accordance with one embodiment the promoter sequence has atotal length of no more than 1.5, 2, 2.5, 3 or 4 kb. In accordance withone embodiment the Zea mays promoter consists of SEQ ID NO: 1 or about a379 bp sequence that has 90, 95 or 99% sequence identity with SEQ IDNO:1.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of SEQ ID NO:1, anon-chlorophyll a/b transgene and a 3′-UTR, wherein SEQ ID NO:1 isoperably linked to the 5′ end of the non-chlorophyll a/b transgene andthe 3′-UTR is operably linked to the 3′ end of the non-chlorophyll a/btransgene. In a further embodiment the 3′ untranslated sequencecomprises SEQ ID NO:6 or a sequence that has 90, 95, 99 or 100% sequenceidentity with SEQ ID NO:6. In a further embodiment the 3′ untranslatedsequence consists of SEQ ID NO:6, or about a 295 bp sequence that has90, 95, or 99% sequence identity with SEQ ID NO:6.

In accordance with one embodiment the nucleic acid vector furthercomprises a sequence encoding a selectable maker. In accordance with oneembodiment the recombinant gene cassette is operably linked to anAgrobacterium T-DNA border. In accordance with one embodiment therecombinant gene cassette further comprises a first and second T-DNAborder, wherein first T-DNA border is operably linked to one end of thegene construct, and said second T-DNA border is operably linked to theother end of the gene construct. The first and second AgrobacteriumT-DNA borders can be independently selected from T-DNA border sequencesoriginating from bacterial strains selected from the group consisting ofa nopaline synthesizing Agrobacterium T-DNA border, an ocotopinesynthesizing Agrobacterium T-DNA border, a succinamopine synthesizingAgrobacterium T-DNA border, or any combination thereof. In oneembodiment an Agrobacterium strain selected from the group consisting ofa nopaline synthesizing strain, a mannopine synthesizing strain, asuccinamopine synthesizing strain, or an octopine synthesizing strain isprovided, wherein said strain comprises a plasmid wherein the plasmidcomprises a transgene operably linked to a sequence selected from SEQ IDNO:6 or a sequence having 90, 95, or 99% sequence identity with SEQ IDNO:6.

Transgenes of interest and suitable for use in the present disclosedconstructs include, but are not limited to, coding sequences that confer(1) resistance to pests or disease, (2) resistance to herbicides, and(3) value added traits as disclosed in WO2013116700 (DGT-28),US20110107455 (DSM-2), U.S. Pat. No. 8,283,522 (AAD-12); U.S. Pat. No.7,838,733 (AAD-1); U.S. Pat. Nos. 5,188,960; 5,691,308; 6,096,708; and6,573,240 (Cry1F); U.S. Pat. Nos. 6,114,138; 5,710,020; and 6,251,656(Cry1Ac); U.S. Pat. Nos. 6,127,180; 6,624,145 and 6,340,593 (Cry34Ab1);U.S. Pat. Nos. 6,083,499; 6,548,291 and 6,340,593 (Cry35Ab1), thedisclosures of which are incorporated herein. In accordance with oneembodiment the transgene encodes a selectable marker or a gene productconferring insecticidal resistance, herbicide tolerance, nitrogen useefficiency, water use efficiency, or nutritional quality.

In an embodiment, a cell or plant is provided comprising a geneexpression cassette as disclosed herein. In an embodiment, a cell orplant comprises a vector comprising a gene expression cassette asdisclosed herein. In an embodiment, a vector can be a plasmid, a cosmid,a bacterial artificial chromosome (BAC), a bacteriophage, or a virus.Thereby, a cell or plant comprising a gene expression cassette asdisclosed herein is a transgenic cell or transgenic plant, respectively.In an embodiment, a transgenic plant can be a monocotyledonous plant. Inan embodiment, a transgenic monocotyledonous plant can be, but is notlimited to maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane,and millet. In an embodiment, a transgenic plant can be a dicotyledonousplant. In an embodiment, a transgenic dicotyledonous plant can be, butis not limited to soybean, cotton, sunflower, and canola. An embodimentalso includes a transgenic seed from a transgenic plant as disclosedherein.

In an embodiment, a gene expression cassette includes two or moretransgenes. The two or more transgenes may not be operably linked to apromoter, intron, or 5′-UTR or 3′-UTR as disclosed herein. In anembodiment, a gene expression cassette includes one or more transgenes.In an embodiment with one or more transgenes, at least one transgene isoperably linked to a promoter, intron, 5′-UTR, or 3′-UTR or the subjectdisclosure.

Selectable Markers:

Various selectable markers also described as reporter genes can beincorporated into a chosen expression vector to allow for identificationand selection of transformed plants (“transformants”). Many methods areavailable to confirm expression of selectable markers in transformedplants, including for example DNA sequencing and PCR (polymerase chainreaction), Southern blotting, Northern blotting, immunological methodsfor detection of a protein expressed from the vector, e g., precipitatedprotein that mediates phosphinothricin resistance, or visual observationof other proteins such as reporter genes encoding β-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), red fluorescent protein (RFP), β-galactosidase, alkalinephosphatase, and the like (See Sambrook, et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y., 2001,the content of which is incorporated herein by reference in itsentirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO)and hygromycin phosphotransferase (HPT) as well as genes conferringresistance to herbicidal compounds. Herbicide resistance genes generallycode for a modified target protein insensitive to the herbicide or foran enzyme that degrades or detoxifies the herbicide in the plant beforeit can act. For example, resistance to glyphosate has been obtained byusing genes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or DGT-28. Genes andmutants for EPSPS are well known, and further described below.Resistance to glufosinate ammonium, bromoxynil, and2,4-dichlorophenoxyacetate (2,4-D) have been obtained by using bacterialgenes encoding pat or DSM-2, a nitrilase, an aad-1 or an aad-12 gene,which detoxifies the respective herbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar genes from Streptomyces species,including Streptomyces hygroscopicus and Streptomyces viridichromogenes,and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). Exemplary genes conferring resistance tocyclohexanediones and/or aryloxyphenoxypropanoic acid (includingHaloxyfop, Diclofop, Fenoxyprop, Fluazifop, Quizalofop) include genes ofacetyl coenzyme A carboxylase (ACCase)-Acc1-S1, Acc1-S2 and Acc1-S3. Inan embodiment, herbicides can inhibit photosynthesis, including triazine(psbA and 1s+ genes) or benzonitrile (nitrilase gene).

In an embodiment, selectable marker genes include, but are not limitedto genes encoding: neomycin phosphotransferase II; cyanamide hydratase;aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA).

An embodiment also includes genes encoding resistance to:chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil;glyphosate; and phosphinothricin.

The above list of selectable marker genes is not meant to be limiting.Any reporter or selectable marker gene are encompassed by the presentinvention.

Selectable marker genes are synthesized for optimal expression in aplant. For example, in an embodiment, a coding sequence of a gene hasbeen modified by codon optimization to enhance expression in plants. Aselectable marker gene can be optimized for expression in a particularplant species or alternatively can be modified for optimal expression indicotyledonous or monocotyledonous plants. Plant preferred codons may bedetermined from the codons of highest frequency in the proteinsexpressed in the largest amount in the particular plant species ofinterest. In an embodiment, a selectable marker gene is designed to beexpressed in plants at a higher level resulting in higher transformationefficiency. Methods for plant optimization of genes are well known.Guidance regarding the optimization and production of synthetic DNAsequences can be found in, for example, WO2013016546, WO2011146524,WO1997013402, U.S. Pat. No. 6,166,302, and U.S. Pat. No. 5,380,831,herein incorporated by reference. Transformation;

Suitable methods for transformation of plants include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253);micro-projectile bombardment (see, e.g., U.S. Pat. Nos. 5,015,580,5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865);Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos.5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); andprotoplast transformation (see, e.g., U.S. Pat. No. 5,508,184). Thesemethods may be used to stably transform or transiently transform aplant.

A DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as agitation with silicon carbidefibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-73). Alternatively, the DNA construct can beintroduced into the plant cell via nanoparticle transformation (see,e.g., US Patent Publication No. 20090104700, which is incorporatedherein by reference in its entirety).

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

Through the application of transformation techniques, cells of virtuallyany plant species may be stably transformed, and these cells may bedeveloped into transgenic plants by well-known techniques. For example,techniques that may be particularly useful in the context of cottontransformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135,5,004,863, and 6,624,344; techniques for transforming Brassica plants inparticular are described, for example, in U.S. Pat. No. 5,750,871;techniques for transforming soy bean are described, for example, in U.S.Pat. No. 6,384,301; and techniques for transforming maize are described,for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, andInternational PCT Publication WO 95/06722.

After effecting delivery of an exogenous nucleic acid to a recipientcell, a transformed cell is generally identified for further culturingand plant regeneration. In order to improve the ability to identifytransformants, one may desire to employ a selectable marker gene withthe transformation vector used to generate the transformant. In anillustrative embodiment, a transformed cell population can be assayed byexposing the cells to a selective agent or agents, or the cells can bescreened for the desired marker gene trait.

Cells that survive exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. In an embodiment, any suitable planttissue culture media may be modified by including further substances,such as growth regulators. Tissue may be maintained on a basic mediawith growth regulators until sufficient tissue is available to beginplant regeneration efforts, or following repeated rounds of manualselection, until the morphology of the tissue is suitable forregeneration (e.g., at least 2 weeks), then transferred to mediaconducive to shoot formation. Cultures are transferred periodicallyuntil sufficient shoot formation has occurred. Once shoots are formed,they are transferred to media conducive to root formation. Oncesufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

To confirm the presence of a desired nucleic acid comprising constructsprovided in regenerating plants, a variety of assays may be performed.Such assays may include: molecular biological assays, such as Southernand northern blotting and PCR; biochemical assays, such as detecting thepresence of a protein product, e.g., by immunological means (ELISA,western blots, and/or LC-MS MS spectrophotometry) or by enzymaticfunction; plant part assays, such as leaf or root assays; and/oranalysis of the phenotype of the whole regenerated plant.

Transgenic events may be screened, for example, by PCR amplificationusing, e.g., oligonucleotide primers specific for nucleic acid moleculesof interest. PCR genotyping is understood to include, but not be limitedto, polymerase-chain reaction (PCR) amplification of genomic DNA derivedfrom isolated host plant callus tissue predicted to contain a nucleicacid molecule of interest integrated into the genome, followed bystandard cloning and sequence analysis of PCR amplification products.Methods of PCR genotyping have been well described (see, e.g., Rios etal. (2002) Plant J. 32:243-53), and may be applied to genomic DNAderived from any plant species or tissue type, including cell cultures.Combinations of oligonucleotide primers that bind to both targetsequence and introduced sequence may be used sequentially or multiplexedin PCR amplification reactions. Oligonucleotide primers designed toanneal to the target site, introduced nucleic acid sequences, and/orcombinations of the two may be produced. Thus, PCR genotyping strategiesmay include, for example and without limitation: amplification ofspecific sequences in the plant genome; amplification of multiplespecific sequences in the plant genome; amplification of non-specificsequences in the plant genome; and combinations of any of the foregoing.One skilled in the art may devise additional combinations of primers andamplification reactions to interrogate the genome. For example, a set offorward and reverse oligonucleotide primers may be designed to anneal tonucleic acid sequence(s) specific for the target outside the boundariesof the introduced nucleic acid sequence.

Forward and reverse oligonucleotide primers may be designed to annealspecifically to an introduced nucleic acid molecule, for example, at asequence corresponding to a coding region within a nucleotide sequenceof interest comprised therein, or other parts of the nucleic acidmolecule. Primers may be used in conjunction with primers describedherein. Oligonucleotide primers may be synthesized according to adesired sequence and are commercially available (e.g., from IntegratedDNA Technologies, Inc., Coralville, Iowa). Amplification may be followedby cloning and sequencing, or by direct sequence analysis ofamplification products. In an embodiment, oligonucleotide primersspecific for the gene target are employed in PCR amplifications. Methodof Expressing a Transgene

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a Zea mays promoter operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a Zea mays 5′-UTR operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant comprising growing a plant comprising a Zea mays intron operablylinked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a Zea mays promoter, a Zea mays 5′-UTR, and a Zea mays intronoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a Zea mays 3′-UTR operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a Zea mays promoter operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene in a plant tissue or plant cell comprising culturing a planttissue or plant cell comprising a Zea mays 5′-UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene in a plant tissue or plant cell comprising culturing aplant tissue or plant cell comprising a Zea mays intron operably linkedto at least one transgene. In an embodiment, a method of expressing atleast one transgene in a plant tissue or plant cell comprising culturinga plant tissue or plant cell comprising a Zea mays promoter, a Zea mays5′-UTR, and a Zea mays intron operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant tissue or plant cell comprising culturing a plant tissue or plantcell comprising a Zea mays 3′-UTR operably linked to at least onetransgene.

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a Zea mays promoter operably linked to at least onetransgene. In one embodiment the Zea mays promoter consists of asequence selected from SEQ ID NO:1, or a sequence that has 90, 95 or 99%sequence identity with a sequence selected from SEQ ID NO:1. In anembodiment, a method of expressing at least one transgene in a plantcomprises growing a plant comprising a gene expression cassettecomprising a Zea mays intron operably linked to at least one transgene.In an embodiment, a method of expressing at least one transgene in aplant linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant comprises growing a plantcomprising a gene expression cassette comprising a Zea mays promoter, aZea mays 5′-UTR, and a Zea mays intron operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene in a plant comprises growing a plant comprising a geneexpression cassette comprising a Zea mays 3′-UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene in a plant tissue or plant cell comprises culturing aplant tissue or plant cell comprising a gene expression cassette a Zeamays promoter operably linked to at least one transgene. In anembodiment, a method of expressing at least one transgene in a planttissue or plant cell comprises culturing a plant tissue or plant cellcomprising a gene expression cassette a Zea mays intron operably linkedto at least one transgene. In an embodiment, a method of expressing atleast one transgene in a plant tissue or plant cell comprises culturinga plant tissue or plant cell comprising a gene expression cassette a Zeamays 5′-UTR operably linked to at least one transgene. In an embodiment,a method of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette a Zea mays promoter, a Zea mays 5′-UTR, and a Zeamays intron operably linked to at least one transgene. In an embodiment,a method of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette comprising a Zea mays 3′-UTR operably linked to atleast one transgene.

Transgenic Plants;

In an embodiment, a plant, plant tissue, or plant cell comprises a Zeamays promoter. In an embodiment, a Zea mays promoter can be a promoterof SEQ ID NO:1 or a promoter with at least 90%, 95%, or 99% sequenceidentity with SEQ ID NO:1. In an embodiment, a plant, plant tissue, orplant cell comprises a gene expression cassette comprises a promoter,wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1,wherein the promoter is operably linked to a non-chlorophyll a/btransgene. In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprising a sequence selected fromSEQ ID NO:1, or a sequence that has 90, 95 or 995 sequence identity witha sequence selected from SEQ ID NO:1 that is operably linked to anon-chlorophyll a/b transgene. In an illustrative embodiment, a plant,plant tissue, or plant cell comprises a gene expression cassettecomprising a Zea mays promoter that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a 3′-UTR. In an embodiment, a plant,plant tissue, or plant cell comprises a gene expression cassettecomprising a Zea mays 3′-UTR.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising an intron, wherein the intron is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:2 or SEQ ID NO:3. In anembodiment, a gene expression cassette comprises a Zea mays intron thatis operably linked to a promoter, wherein the promoter is a Zea mayspromoter of SEQ ID NO:1, or a promoter that originates from a plant(e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava veinmosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciensdelta mas). In an embodiment, a plant, plant tissue, or plant cellcomprises a gene expression cassette comprising a Zea mays intron thatis operably linked to a transgene. In an illustrative embodiment, aplant, plant tissue, or plant cell comprising a gene expression cassettecomprising a Zea mays intron that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a 5′-UTR, wherein the 5′-UTR is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identical to SEQ ID NO:4. In an embodiment, a geneexpression cassette comprises a Zea mays 5′-UTR that is operably linkedto a promoter, wherein the promoter is a Zea mays promoter of SEQ IDNO:1, or a promoter that originates from a plant (e.g., Zea maysubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic viruspromoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). Inan embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Zea mays 5′-UTR that is operably linkedto a transgene. In an illustrative embodiment, a plant, plant tissue, orplant cell comprising a gene expression cassette comprising a Zea mays5′-UTR that is operably linked to a transgene, wherein the transgene canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Zea mays promoter and a Zea mays3′-UTR. In an embodiment, a plant, plant tissue, or plant cell comprisesa gene expression cassette comprising a) a promoter, wherein thepromoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1 and b) a3′-UTR, wherein the 3′-UTR is at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ IDNO:6.

In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a Zea mays promoter, Zea mays 5′-UTR, Zeamays intron, and a Zea mays 3′-UTR that are operably linked to atransgene. The promoter, intron, 5′-UTR, and 3′-UTR can be operablylinked to different transgenes within a gene expression cassette when agene expression cassette includes two or more transgenes. In anillustrative embodiment, a gene expression cassette comprises a Zea mayspromoter that is operably linked to a transgene, wherein the transgenecan be an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, aselectable marker transgene, or combinations thereof. In an illustrativeembodiment, a gene expression cassette comprises a Zea mays intron thatis operably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a selectablemarker transgene, or combinations thereof. In an embodiment, a geneexpression cassette comprises a Zea mays intron that is operably linkedto a promoter, wherein the promoter is a Zea mays promoter of SEQ IDNO:1, or a promoter that originates from a plant (e.g., Zea maysubiquitin 1 promoter), a virus (e.g., Cassava vein mosaic viruspromoter) or a bacteria (e.g., Agrobacterium tumefaciens delta mas). Inan illustrative embodiment, a gene expression cassette comprises a Zeamays 5′-UTR that is operably linked to a transgene, wherein thetransgene can be an insecticidal resistance transgene, an herbicidetolerance transgene, a nitrogen use efficiency transgene, a water useefficiency transgene, a nutritional quality transgene, a DNA bindingtransgene, a selectable marker transgene, or combinations thereof. In anembodiment, a gene expression cassette comprises a Zea mays 5′-UTR thatis operably linked to a promoter, wherein the promoter is a Zea mayspromoter of SEQ ID NO:1, or a promoter that originates from a plant(e.g., Zea mays ubiquitin 1 promoter), a virus (e.g., Cassava veinmosaic virus promoter) or a bacteria (e.g., Agrobacterium tumefaciensdelta mas). In an illustrative embodiment, a gene expression cassettecomprises a Zea mays 3′-UTR that is operably linked to a transgene,wherein the 3′-UTR can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In an embodiment, transgene expression using methods described herein isspecific to a plant's leaf tissues. In an embodiment, transgeneexpression includes more than one transgene expressed in the plant'sleaf tissues. In an embodiment, a method of growing a transgenic plantas described herein includes leaf-specific transgene expression. In anembodiment, a method of expressing a transgene in a plant tissue orplant cell includes leaf-specific tissues and root-specific cells. In anembodiment, the leaf-specific expression includes maize leaf-specificexpression.

In a further embodiment, transgene expression using methods describedherein is expressed within above ground plant tissues (e.g., aboveground plant tissues include leaf, husk, stem, and silk). In anembodiment, transgene expression includes more than one transgeneexpressed in above ground plant tissues. In an embodiment, a method ofgrowing a transgenic plant as described herein includes above groundplant tissues transgene expression. In an embodiment, a method ofexpressing a transgene in a plant tissue or plant cell above groundplant tissues and above ground plant cells. In an embodiment, the aboveground plant tissue expression includes maize above ground plant tissueexpression.

In an embodiment, a plant, plant tissue, or plant cell comprises avector comprising a Zea mays promoter, Zea mays 5′-UTR, Zea mays intron,and/or Zea mays 3′-UTR as disclosed herein. In an embodiment, a plant,plant tissue, or plant cell comprises a vector comprising a Zea mayspromoter, Zea mays 5′-UTR, Zea mays intron, and/or Zea mays 3′-UTR asdisclosed herein operably linked to a non-chlorophyll a/b transgene. Inan embodiment, a plant, plant tissue, or plant cell comprises a vectorcomprising a gene expression cassette as disclosed herein. In anembodiment, a vector can be a plasmid, a cosmid, a bacterial artificialchromosome (BAC), a bacteriophage, or a virus.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises anon-endogenous Zea mays derived promoter sequence operably linked to atransgene, wherein the Zea mays promoter sequence comprises a sequenceof SEQ ID NO:1 or a sequence having 90. 95, 98 or 99% sequence identitywith SEQ ID NO:1. In one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises SEQID NO: 1, or a sequence that has 90% sequence identity with SEQ ID NO: 1operably linked to a non-chlorophyll a/b transgene. In one embodimentthe plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or a cell or tissue derived from a dicotyledonousor monocotyledonous plant. In one embodiment the plant is selected fromthe group consisting of maize, wheat, rice, sorghum, oats, rye, bananas,sugar cane, soybean, cotton, sunflower, and canola. In one embodimentthe plant is Zea mays.

In one embodiment a plant, plant tissue, or plant cell is providedcomprising SEQ ID NO:1, or a sequence that has 90, 95, 98 or 99%sequence identity with SEQ ID NO:1, operably linked to a transgene. Inaccordance with one embodiment the plant, plant tissue, or plant cell isa dicotyledonous or monocotyledonous plant or plant cell or tissuederived from a dicotyledonous or monocotyledonous plant. In oneembodiment the plant is selected from the group consisting of maize,wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton,sunflower, and canola. In one embodiment the plant is Zea mays. Inaccordance with one embodiment the promoter sequence operably linked toa transgene is incorporated into the genome of the plant, plant tissue,or plant cell. In one embodiment the plant, plant tissue, or plant cellfurther comprises a 5′ untranslated sequence comprising SEQ ID NO:4 or asequence that has 90% sequence identity with SEQ ID NO:4, wherein the 5′untranslated sequence is inserted between, and operably linked to, saidpromoter and said transgene. In a further embodiment the plant, planttissue, or plant cell further comprises an intron sequence insertedafter the 5′ untranslated sequence. In one embodiment the intronsequence is an intron sequence isolated from a chlorophyll a/b gene ofZea mays. In one embodiment the intron sequence comprises or consists ofSEQ ID NO:2 or SEQ ID NO:3.

In one embodiment a plant, plant tissue, or plant cell is provided thatcomprises SEQ ID NO:1, or a sequence that has 90. 95, 98 or 99% sequenceidentity with SEQ ID NO:1, operably linked to the 5′ end of a transgeneand a 3′ untranslated sequence comprising SEQ ID NO:6 or a sequence thathas 90% sequence identity with SEQ ID NO:6, wherein the 3′ untranslatedsequence is operably linked to said transgene. In accordance with oneembodiment the plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or is a plant issue or cell derived from adicotyledonous or monocotyledonous plant. In one embodiment the plant isselected from the group consisting of maize, wheat, rice, sorghum, oats,rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. In oneembodiment the plant is Zea mays. In accordance with one embodiment thepromoter sequence operably linked to a transgene is incorporated intothe genome of the plant, plant tissue, or plant cell. In one embodimentthe plant, plant tissue, or plant cell further comprises a 5′untranslated sequence comprising SEQ ID NO:4 or a sequence that has 90%sequence identity with SEQ ID NO:4, wherein the 5′ untranslated sequenceis inserted between, and operably linked to, said promoter and saidtransgene. In a further embodiment the plant, plant tissue, or plantcell further comprises an intron sequence inserted after the 5′untranslated sequence. In one embodiment the intron sequence is anintron sequence isolated from a chlorophyll a/b gene of Zea mays. In oneembodiment the 5′ 5′ untranslated sequence consists of SEQ ID NO:4.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a monocotyledonous plant. Themonocotyledonous plant, plant tissue, or plant cell can be, but notlimited to corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids,bamboo, banana, cattails, lilies, oat, onion, millet, and triticale.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a dicotyledonous plant. Thedicotyledonous plant, plant tissue, or plant cell can be, but notlimited to rapeseed, canola, Indian mustard, Ethiopian mustard, soybean,sunflower, and cotton.

With regard to the production of genetically modified plants, methodsfor the genetic engineering of plants are well known in the art. Forinstance, numerous methods for plant transformation have been developed,including biological and physical transformation protocols fordicotyledonous plants as well as monocotyledonous plants (e.g.,Goto-Fumiyuki et al., Nature Biotech 17:282-286 (1999); Miki et al.,Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. andThompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993)). Inaddition, vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available, for example, inGruber et al., Methods in Plant Molecular Biology and Biotechnology,Glick, B. R. and Thompson, J. E. Eds., CRC Press, Inc., Boca Raton, pp.89-119 (1993).

One of skill in the art will recognize that after the exogenous sequenceis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed cells can also be identified by screening for theactivities of any visible marker genes (e.g., the yfp, gfp,β-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid vectors. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. Thesemethods include but are not limited to: 1) Southern analysis or PCRamplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, S1 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) Next GenerationSequencing (NGS) analysis; 5) protein gel electrophoresis, Western blottechniques, immunoprecipitation, or enzyme-linked immunoassay (ELISA),where the gene construct products are proteins. Additional techniques,such as in situ hybridization, enzyme staining, and immunostaining, alsomay be used to detect the presence or expression of the recombinantconstruct in specific plant organs and tissues. The methods for doingall these assays are well known to those skilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, Northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the mRNA is presentor the amount of mRNA has increased, it can be assumed that thecorresponding transgene is being expressed. Other methods of measuringgene and/or encoded polypeptide activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of polypeptide expressed can bemeasured immunochemically, i.e., ELISA, RIA, EIA and other antibodybased assays well known to those of skill in the art, such as byelectrophoretic detection assays (either with staining or Westernblotting). As one non-limiting example, the detection of the AAD-1(aryloxyalkanoate dioxygenase; see WO 2005/107437) and PAT(phosphinothricin-N-acetyl-transferase), proteins using an ELISA assayis described in U.S. Patent Publication No. 20090093366 which is hereinincorporated by reference in its entirety. The transgene may beselectively expressed in some cell types or tissues of the plant or atsome developmental stages, or the transgene may be expressed insubstantially all plant tissues, substantially along its entire lifecycle. However, any combinatorial expression mode is also applicable.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein saidprogeny, clone, cell line or cell has the transgene or gene construct.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

EXAMPLES Example 1 Identification of High Expressing Regulatory Elements

Novel Zea mays regulatory elements were identified via a transcriptionalprofiling approach by using next generation sequencing (NGS). Theseregulatory elements were then isolated, and cloned to characterize theexpression profile of the regulatory elements for use in transgenicplants. Transgenic maize lines stably transformed with a cry34Ab1reporter gene isolated from Bacillus thuringiensis, a phiyfp reportergene, and an aad-1 selectable marker gene were produced and thetransgene expression levels and tissue specificity was assessed. As suchnovel Zea mays regulatory elements were identified and characterized.Disclosed for the first time are promoter and 3′-UTR regulatory elementsfor use in gene expression constructs.

Maize tissues were obtained from plants grown to different stages ofplant growth and development for transcriptional profiling in order toidentify and select regulatory elements of native maize genes withdesired expression profiles for use in gene expression constructs. Forexample, tissue samples from 3 stages of leaf (V4 (duplicate), V12 andR3) and root (V4 and V12 nodal and fibrous tissues) development, pollen,silk, cob, immature kernel, husk and stem (V4 and R1) were collected.Total mRNA was isolated from all of the above described tissues and highquality mRNA in desired quantities were obtained.

cDNA libraries were prepared from each of the mRNA samples andhigh-throughput sequencing was completed using an Illumina HiSeq® 2000(Illumina Inc., San Diego, Calif.). In addition, the Illumina TruSeq®RNA sample preparation kit was used according to the manufacturer'srecommended protocol for RNAseq sample preparation. In brief, 5 μg oftotal RNA was purified using poly-T oligo-attached magnetic beadsfollowed by fragmentation into smaller pieces (about 200 bp averagelength) using divalent cations under high temperature. SuperScript® IIreverse transcriptase and random primers were then used to copy thefragmented mRNA into first strand cDNA. The cDNA was further convertedinto double stranded cDNA (ds cDNA) using DNA polymerase I and RNase H.The double stranded cDNA fragments then went through end repair,A-tailing, and then ligation to indexed Illumina paired-end (PE)adapters. Lastly, library products were cleaned up and enriched with 15cycles of PCR and purified. The enriched libraries were normalized to aconcentration of 2 nM, denatured with sodium hydroxide, and diluted to12 pM in hybridization buffer for loading onto a single lane of a HiSeq®flow cell. Cluster generation, primer hybridization and sequencingreactions were carried out according to Illumina's recommended protocol.

The sequencing reads were then filtered to remove low quality reads.About 99.9% of the sequencing reads were retained after filtering. Thesequencing reads were aligned to the annotated Zea mays c.v. B73 genomeavailable in the maizeGDB. Sequencing reads that mapped onto the maizegenome at more than one locus were discarded to avoid confusion inidentification of the high expressing genes and their furthercharacterization. This step led to alignment of >70% sequencing readsfrom each of the samples to the maize genome. The quantitative geneexpression unit of fragments per kilobase of exon per million fragmentsmapped or FPKM values were used to rank genes for stable transformationtesting that matched a desirable expression pattern for use in geneexpression constructs. Highly expressed genes in maize were prioritizedfor testing in stable transgenic lines (FIG. 1).

Example 2 Selection of Novel Regulatory Elements from Zea mays Sequence

The promoter, intron, 5′-UTR, and 3′-UTR sequences were extracted from aZea mays gene sequence that ranked highly for expression through thetranscriptional profiling approach. The native sequence of the Zea maysgene, from the Zea may c.v. B73 genome, is provided as SEQ ID NO:7. The379 bp Zea mays promoter sequence (SEQ ID NO:1) is provided. In additionto the 119 bp Zea mays intron sequence (SEQ ID NO:2), and the 91 bp Zeamays intron (2) sequence (SEQ ID NO:3) are provided as fragments makingup SEQ ID NO:7. Furthermore, the 152 bp Zea mays 5′-UTR sequence (SEQ IDNO:4) is provided. Finally, the 295 bp Zea mays 3′-UTR sequence (SEQ IDNO:6) is also provided.

Example 3 Construct Design

The DNA elements were synthesized and cloned into entry vectors. The Zeamays promoter, intron, and 5′-UTR (SEQ ID NO:5), cry34Ab1 (reporter genefrom B. thuringiensis), and the Solanum tuberosum protease inhibitorgene II 3′ UTR (StPinII 3′-UTR v2; An et al., (1989) Plant Cell 1;115-22) were amplified with primers containing a minimum 15 bpoverlapping homology to their flanking DNA element within the donorconstruct. All fragments were gel purified. All three fragments alongwith an entry vector backbone, pENTR11, were assembled in a directionalorder through a Geneart® Seamless cloning reaction (Invitrogen,Carlsbad, Calif.). The resulting gene expression cassette contained thecry34Ab1 transgene operably linked to the 3′ end of the Zea mayspromoter, intron, and 5′-UTR (SEQ ID NO:5). A Gateway® LR Clonase®(Invitrogen) reaction was then performed with the resulting entryplasmid, pDAB114404, and a destination vector, pDAB104153, leading to afinal expression vector, pDAB114411. The destination vector contained aselectable marker cassette comprised of an aad-1 gene driven by the Zeamays ubiquitin-1 promoter (Christensen et al., (1992) Plant MolecularBiology 18; 675-689) and terminated by a maize lipase 3′-UTR (U.S. Pat.No. 7,179,902). The resulting construct, pDAB114411 is a heterologousexpression construct that contains an aad-1 gene expression cassette anda cry34Ab1 gene expression cassette (FIG. 2).

A second entry vector, pDAB116024, cassette was assembled with Geneart®Seamless cloning reaction by replacing the cry34Ab1 gene with a phiYFPreporter gene (Shagin et al., (2004) Mol Biol Evol 21; 841-50)containing a Solanum tuberosum LS1 intron (St-LS1; Vancanneyt G F etal., (1990) Mol Gen Genet 220(2):245-250) and replacing the StPinII3′-UTR with the novel 295 bp 3′-UTR sequence (SEQ ID NO:6) obtained fromZea mays. Both the promoter, intron, and 5′-UTR (SEQ ID NO:5) and 3′-UTR(SEQ ID NO:6) elements were derived from the same native gene of Zeamays. The resulting gene expression cassette contained the phiYFPtransgene operably linked to the 3′ end of the Zea mays promoter,intron, and 5′-UTR (SEQ ID NO:5). Following a Gateway reaction with adestination vector, pDAB104153, a final expression construct,pDAB116038, was assembled. The destination vector contained a selectablemarker cassette comprised of an aad-1 gene driven by the Zea maysubiquitin-1 promoter (Christensen et al., (1992) Plant Molecular Biology18; 675-689) and terminated by a maize lipase 3′-UTR (U.S. Pat. No.7,179,902). The resulting construct, pDAB116038 is a heterologousexpression construct that contains an aad-1 gene expression cassette anda phiYFP gene expression construct (FIG. 3).

A negative control construct, pDAB101556, was assembled containing ayellow fluorescence protein (YFP) reporter gene instead of the cry34Ab1gene (FIG. 4) and the same aad-1 expression cassette as present inpDAB114411. A positive control construct, pDAB108746, was builtcomprised of the Zea mays ubiquitin-1 promoter and Solanum tuberosumprotease inhibitor gene II 3′ UTR (StPinII 3′-UTR v2; An et al., (1989)Plant Cell 1; 115-22) controlling the expression of the cry34Ab1 gene(FIG. 5). The aad-1 cassette was the same as present in pDAB114411.Another positive control construct, pDAB113121, was built comprised ofthe Zea mays ubiquitin-1 promoter and Solanum tuberosum proteaseinhibitor gene II 3′ UTR (StPinII 3′-UTR v2; An et al., (1989) PlantCell 1; 115-22) controlling the expression of the phiYFP gene containingan St-LS1 intron (FIG. 6). The aad-1 cassette was the same as present inpDAB116038.

Example 4 Plant Transformation and Molecular Confirmation

Transformation of Agrobacterium tumefaciens:

The binary expression vectors were transformed into Agrobacteriumtumefaciens strain DAt13192 (RecA deficient ternary strain) (Int. Pat.Pub. No. WO2012016222). Bacterial colonies were selected, and binaryplasmid DNA was isolated and confirmed via restriction enzyme digestion.

Agrobacterium Culture Initiation:

Agrobacterium cultures were streaked from glycerol stocks onto ABminimal medium (Gelvin, S., 2006, Agrobacterium Virulence GeneInduction, in Wang, K., ed., Agrobacterium Protocols Second Edition Vol.1, Humana Press, p. 79; made without sucrose and with 5 g/L glucose and15 g/L Bacto™ Agar) and incubated at 20° C. in the dark for 3 days.Agrobacterium cultures were then streaked onto a plate of YEP medium(Gelvin, S., 2006, Agrobacterium Virulence Gene Induction, in Wang, K.,ed., Agrobacterium Protocols Second Edition Vol. 1, Humana Press, p. 79)and incubated at 20° C. in the dark for 1 day.

On the day of an experiment, a mixture of Inoculation medium (2.2 g/L MSsalts, 68.4 g/L sucrose, 36 g/L glucose, 115 mg/L L-proline, 2 mg/Lglycine, 100 mg/L myo-Inositol, 0.05 mg/L nicotinic acid, 0.5 mg/Lpyridoxine HCl, 0.5 mg/L thiamine HCl) and acetosyringone was preparedin a volume appropriate to the size of the experiment. A 1 M stocksolution of acetosyringone in 100% dimethyl sulfoxide was added to theInoculation medium to make a final acetosyringone concentration of 200μM.

For each construct, 1-2 loops of Agrobacterium from the YEP plate weresuspended in 15 ml of the inoculation medium/acetosyringone mixtureinside a sterile, disposable, 50 ml centrifuge tube and the opticaldensity of the solution at 600 nm (O.D.₆₀₀) was measured in aspectrophotometer. The suspension was then diluted down to 0.25-0.35O.D.₆₀₀ using additional Inoculation medium/acetosyringone mixture. Thetube of Agrobacterium suspension was then placed horizontally on aplatform shaker set at about 75 rpm at room temperature for between 1and 4 hours before use.

Maize Transformation:

Experimental constructs were transformed into maize viaAgrobacterium-mediated transformation of immature embryos isolated fromthe inbred line, Zea mays c.v. B104. The method used is similar to thosepublished by Ishida et al., (1996) Nature Biotechnol 14:745-750 andFrame et al., (2006) Plant Cell Rep 25: 1024-1034, but with severalmodifications and improvements to make the method amenable tohigh-throughput transformation. An example of a method used to produce anumber of transgenic events in maize is given in U.S. Pat. App. Pub. No.US 2013/0157369 A1, beginning with the embryo infection andco-cultivation steps. Transfer and establishment of T₀ plants in thegreenhouse:

Transgenic plants were transferred on a regular basis to the greenhouse.Plants were transplanted from Phytatrays™ to small pots (T. O. Plastics,3.5″ SVD, 700022C) filled with growing media (Premier Tech Horticulture,ProMix BX) and covered with humidomes to help acclimate the plants.Plants were placed in a Conviron™ growth chamber (28° C./24° C., 16-hourphotoperiod, 50-70% RH, 200 μmol m⁻² s⁻¹ light intensity) until reachingV3-V4 stage. This aided in acclimating the plants to soil and harshertemperatures. Plants were then moved to the greenhouse (Light ExposureType: Photo or Assimilation; High Light Limit: 1200 μmol m⁻² s⁻¹photosynthetically active radiation (PAR); 16-hour day length; 27° C.Day/24° C. Night) and transplanted from the small pots to 5.5 inch pots.Approximately 1-2 weeks after transplanting to larger pots the plantswere sampled for bioassay. One plant per event was assayed.

Example 5 Molecular Confirmation of Transgenic Plants/Events

Putative transgenic maize plants were sampled at the V2-3 leaf stage fortransgene presence using cry34Ab1 and AAD-1 quantitative PCR assays.Total DNA was extracted from the leaf samples using MagAttract® DNAextraction kit (Qiagen) as per manufacturer's instructions.

To detect the genes-of-interest, gene-specific DNA fragments wereamplified with TaqMan® primer/probe sets containing a FAM-labeledfluorescent probe for the cry34Ab1 gene and a HEX-labeled fluorescentprobe for the endogenous invertase reference gene control. The followingprimers were used for the cry34Ab1 and invertase endogenous referencegene amplifications.

Cry34Ab1 Primers/probes: Forward Primer:  TQ.8v6.1.F: (SEQ ID NO: 8)GCCATACCCTCCAGTTG Reverse Primer:  TQ.8v6.1.R:  (SEQ ID NO: 9)GCCGTTGATGGAGTAGTAGATGG Probe: TQ.8v6.1.MGB.P: (SEQ ID NO: 10)5′-/56-FAM/CCGAATCCAACGGCTTCA/MGB Invertase Primers: Forward Primer:InvertaseF: (SEQ ID NO: 11) TGGCGGACGACGACTTGT Reverse Primer:InvertaseR: (SEQ ID NO: 12) AAAGTTTGGAGGCTGCCGT InvertaseProbe:(SEQ ID NO: 13) 5′-/5HEX/CGAGCAGACCGCCGTGTACTT/3BHQ_1/-3′

Next, the PCR reactions were carried out in a final volume of 10 μlreaction containing 5 μl of Roche LightCycler® 480 Probes Master Mix(Roche Applied Sciences, Indianapolis, Ind.); 0.4 μl each of TQ.8v6.1.F,TQ.8v6.1.R, InvertaseF, and InvertaseR primers from 10 μM stocks to afinal concentration of 400 nM; 0.4 μl each of TQ.8v6.1.MGB.P andInvertase Probes from 5 μM stocks to a final concentration of 200 nM,0.1 μl of 10% polyvinylpyrrolidone (PVP) to final concentration of 0.1%;2 μl of 10 ng/μl genomic DNA and 0.5 μl water. The DNA was amplified ina Roche LightCycler® 480 System under the following conditions: 1 cycleof 95° C. for 10 min; 40 cycles of the following 3-steps: 95° C. for 10seconds; 58° C. for 35 seconds and 72° C. for 1 second, and a finalcycle of 4° C. for 10 seconds. The cry34Ab1 copy number was determinedby comparison of Target (gene of interest)/Reference (Invertase gene)values for unknown samples (output by the LightCycler® 480) toTarget/Reference values of cry34Ab1 copy number controls.

The detection of the AAD-1 gene was carried out as described above forthe cry34Ab1 gene using the invertase endogenous reference gene. TheAAD-1 primer sequences were as follows;

AAD1 Forward Primer: (SEQ ID NO: 14) TGTTCGGTTCCCTCTACCAAAAD1 Reverse Primer: (SEQ ID NO: 15) CAACATCCATCACCTTGACTGA AAD1 Probe: (SEQ ID NO: 16) 5′-FAM/CACAGAACCGTCGCTTCAGCAACA-MGB/BHQ-3′.

The detection of the PhiYFP gene was carried out as described above forthe cry34Ab1 gene using the invertase endogenous reference gene. ThePhiYFP primer sequences were as follows;

PhiYFP v3 Forward Primer: (SEQ ID NO: 17) CGTGTTGGGAAAGAACTTGGA PhiYFP v3 Reverse Primer: (SEQ ID NO: 18) CCGTGGTTGGCTTGGTCTPhiYFP v3 Probe:  (SEQ ID NO: 19) 5′FAM/CACTCCCCACTGCCT/MGB_BHQ_1/3′.

Finally, the T₀ plants containing the gene of interest were sampled atV4-5 for Cry34Ab1, PhiYFP, and AAD-1 leaf ELISA assays. Four leafpunches were sampled. Another set of plants were sampled at V4-5 for theentire root mass for both the protein ELISA assays. Leaf and rootCry34Ab1 (Agdia, Inc., Elkart, Ind.) and AAD-1 (Acadia BioScience) ELISAassays were performed as per the manufacturer's instructions.

The PhiYFP ELISA was completed as follows. Plates were coated withmonoclonal anti-YFP capture antibody (Origene; Rockvile, Md.). Themonoclonal anti-YFP capture antibody was diluted in PBS at aconcentration of 1 μg/ml, and 150 μl of solution was added to the wellsof the plates and incubated overnight at 4° C. Next, the plates werewarmed to room temperature for 20 to 30 minutes. The plates were washedfour times with 350 μl of wash buffer (1×PBS and 0.5% Tween 20). Then200 μl of blocking buffer was aliquoted to the plates, and the plateswere incubated at 37° C. for at least one hour. The plates were washedfour times with 350 μl of wash buffer (1×PBS and 0.5% Tween 20).

Standards of recombinantly expressed PhiYFP (Evrogen; Moscow, Russia)were added to the wells in serial dilutions. The standards wereinitially provided at a concentration of 0.0313 ng/ml to 2 ng/ml. Next,the plates were placed on a shaker and incubated at room temperature.The plates were washed four times with 350 μl of wash buffer (1×PBS and0.5% Tween 20). The primary rabbit anti-PhiYFP polyclonal antibody(Evrogen: Moscow, Russia) was reduced in concentration to 1 μg/ml andadded to the plates. Next, the plates were placed on a shaker andincubated at room temperature. The plates were washed four times with350 μl of wash buffer (1×PBS and 0.5% Tween 20). The secondaryanti-rabbit antibody horseradish peroxidase (Pierce; Rockford, Ill.) wasadded to the plates. Next, the plates were placed on a shaker andincubated at room temperature. The plates were washed four times with350 μl of wash buffer (1×PBS and 0.5% Tween 20). Finally, Pierce 1 StepUltra TMB ELISA, substrate for horseradish peroxidase labeled antibody,was added to the wells and gently shaken. The results were quantitatedwith a spectrophotometer.

The Cry34Ab1 leaf ELISA assays were expressed as ng/cm², while the rootELISA results were expressed as parts per million (or ng protein per mgtotal plant protein). Total root protein assays were carried out withthe Bradford detection method as per the manufacturer's instructions.

T₀ plants were selfed and crossed to Zea mays c.v. B104 non-transgenictransformation lines to obtain T₁ seed. Five-six transgenic lines orevents of each of the test regulatory element constructs were advancedfor T₁ protein and RNA gene expression studies and then to T₂ seedproduction. Accordingly, 30-40 T₁ seed of each of the events were sown;seedlings were sprayed with AssureII® at the V2-3 stage of developmentto kill non-transgenic segregants. The transgenic plants were sampled atmultiple stages of plant development for Cry34Ab1, PhiYFP, and AAD-1ELISA as follows: leaf (V4, V12 and R3); root (V4 and R1); stem (R1);pollen (R1); silk (R1); husk (R3); immature kernel (R3); and cob (R3).All tissues were isolated and placed in tubes embedded in dry ice; whichwere then transferred to −80° C. Frozen tissues were lyophilized priorto protein extraction for ELISA.

Putative transgenic T₁ plants containing cry34Ab1, PhiYFP and AAD-1transgenes were sampled at V4-5 for the leaf ELISA assays. Four leafpunches were sampled. The leaf punches were placed into a tube and asingle ⅛″ stainless steel bead (Hoover Precision Products, Cumming, Ga.,USA) was added to each 1.2 ml tube containing 300 μl extraction buffer(1×PBST supplemented with 0.05% Tween 20 and 0.5% BSA). The samples wereprocessed in a Genogrinder™ (SPEX SamplePrep, Metuchen, N.J.) at 1,500rpm for 4 minutes. The samples were centrifuged at 4,000 rpm for 2minutes in a Sorvall Legend XFR™ centrifuge. Next, an additional 300 μlof extraction buffer was added and the samples were processed once morein a Genogrinder™ at 1,500 rpm for 2 minutes. The samples werecentrifuged once more at 4,000 rpm for 7 minutes. Finally, thesupernatant was collected and ELISA assays were completed at differentdilutions along with the protein standards using the commerciallyavailable Cry34Ab1 (Agdia, Inc.) and AAD-1 (Acadia BioScience, LLC)ELISA assay kits, per the manufacturer's instructions.

Protein extraction for various tissue type ELISAs was carried out bygrinding the lyophilized tissue in a paint shaker for 30 seconds. Fortissues needing further grinding, the grinding step was repeated foranother 30 seconds. Garnet powder was added to cover the curved portionat the bottom of the tube. The coarsely ground tissue was transferred to2 ml tubes and filled up to the 0.5 ml mark. One ceramic ball was addedto each tube, as was 0.6 ml of the partial extraction buffer (200 μl ofprotease inhibitor cocktail, 200 μl of 500 mM EDTA, 15.5 mg DTT powderand PBST to 20 ml). All of the tubes were kept on ice for 10 minutes.The cold tubes were transferred to the 2 ml holder of the Genogrinder®.The samples were ground twice for 30 seconds with a 5 minute cooling onice in between. Next, 40 μl of 10% Tween®-20 and 300 μl extractionbuffer were added to the samples. The samples were ground for another 30seconds with 5 minutes of cooling in between. Finally, each sample wascentrifuged at 13,000 rpm for 7 minutes, and the supernatant wascarefully transferred to a new tube to collect the extract. The extractwas re-suspended in the extraction buffer and was diluted as needed forELISA assays.

Example 6 T₀ Transgenic Plant Expression Screening

The ELISA results indicated that the regulatory elements isolated fromZea mays drove leaf tissue-preferred expression of cry34Ab1 in T₀ eventsthat were transformed with construct, pDAB114411. Lower expression ofCry34Ab1 by the Zea mays regulatory elements were observed in the roots(Tables 3 and 4), compared to pDAB108746 positive control, of the T₀events that were transformed with construct, pDAB114411. The eventsproduced from the control construct pDAB108746 expressed Cry34Ab1 inboth leaf and root tissues. There was no Cry34Ab1 leaf expressionobserved in plant events transformed with the control construct,pDAB101556 that did not contain the cry34Ab1 gene. All constructsexpressed the AAD-1 protein in both root and leaf tissues.

The results of the PhiYFP ELISA indicated that T₀ transgenic eventstransformed with construct pDAB116038, which expressed a phiYFPtransgene terminated by the Zea mays 3′-UTR regulatory element of thesame gene as the promoter, produced high levels of PhiYFP protein inleaf tissues. The expression levels of the PhiYFP protein in thepDAB116038 events were higher than the expression levels of PhiYFPprotein produced in transgenic events transformed with a positivecontrol construct pDAB113121, which expressed a phiYFP transgeneterminated by the StPinII 3′-UTR regulatory element (Table 5). Bothconstructs, pDAB116038 and pDAB113121, expressed the AAD-1 protein inboth root and leaf tissues. Furthermore, transgenic pDAB116038 eventsproduced with the Zea mays 3′-UTR resulted in increased expression ofPhiYFP protein as compared to expression of Cry34Ab1 protein producedfrom cry34Ab1 transgenic events transformed with pDAB114411 andpDAB108746, both of which contained a StPinII 3′-UTR.

TABLE 3 T₀ ELISA results showing cry34Ab1 and aad-1 transgene expressionin V4-V6 maize leaves of various construct events. No. of Mean Cry34Ab1Mean AAD-1 Construct Events Cry34Ab1 Standard AAD-1 Standard NameAnalyzed (ng/cm²) Deviation (ng/cm²) Deviation Experiment 1 pDAB101556 60 0 304 229 pDAB108746 5 173 75 247 146 pDAB114411 15 71 41 226 157Experiment 2 pDAB101556 6 0 0 304 229 pDAB108746 18 129 79 173 96pDAB114411 30 64 40 233 151

TABLE 4 T₀ ELISA assay results showing cry34Ab1 and aad-1 transgeneexpression in V4-6 maize roots of various construct events. No. of MeanCry34Ab1 Mean AAD-1 Construct Events Cry34Ab1 Standard AAD-1 StandardName Analyzed (ng/mg) Deviation (ng/mg) Deviation Experiment 1pDAB101556 1 0 0 871 0 pDAB108746 2 970 1243 445 97 pDAB114411 5 72 97399 290 Experiment 2 pDAB101556 2 0 0 1204 798 pDAB108746 3 2938 2653538 304 pDAB114411 1 76 33 1132 592

TABLE 5 T₁ ELISA assay results showing cry34Ab1 and aad-1 transgeneexpression in multiple tissue types of maize in various constructevents. Total Total Mean Mean Construct Tissue events samples Cry34Ab1Standard AAD-1 Standard No. analyzed analyzed analyzed (ng/mg) Deviation(ng/mg) Deviation pDAB101556 Leaf V4 1 13 1 0 497 161 pDAB114411 Leaf V43 31 753 199 1133 688 pDAB114411 Leaf V12 3 12 1010 211 1698 799pDAB114411 Leaf R3 3 9 3260 1068 2133 1031 pDAB101556 Root V4 1 3 4 32722 234 pDAB114411 Root V4 3 8 49 20 3112 2004 pDAB114411 cob 2 10 15667 7431 3056 pDAB114411 silk 2 6 2069 489 12495 3216 pDAB114411 kernel 28 39 32 4823 1854 pDAB114411 stem 2 6 1921 545 12690 4500 pDAB114411husk 2 10 949 297 3337 1130 pDAB114411 pollen 2 5 35 10 2397 1508

TABLE 6 ELISA assay results depicting phiYFP and aad-1 transgeneexpression in T₀ (V4-6) maize leaves when a native 3′ UTR present inpDAB116038 controlled the phiYFP gene expression. No. of Mean YFP MeanAAD-1 Construct Events YFP Standard AAD-1 Standard No. Analyzed (ng/mg)Deviation (ng/mg) Deviation pDAB116038 18 407 286 350 365 pDAB113121 10244 303 91 46

TABLE 7 ELISA assay results depicting phiYFP and aad-1 transgeneexpression in T₁ (V4-6) maize leaves when a native 3′ UTR present inpDAB116038 controlled the phiYFP gene expression. Total Mean Mean LeafTotal events plants PhiYFP AAD1 Sample ID Stage analyzed analyzed(ng/mg) STD (ng/mg) STD pDAB108746 Leaf V4 1 10 6 0 136 66 pDAB116038Leaf V4 3 22 975 113 241 293 pDAB108746 Leaf V12 1 3 6 0 347 63pDAB116038 Leaf V12 3 9 769 268 728 463 pDAB108746 Leaf R3 1 3 6 0 1009218 pDAB 116038 Leaf R3 3 9 185 47 2216 1162

As such novel maize regulatory elements isolated from Zea mays wereidentified and characterized. Disclosed for the first time are promoter(SEQ ID NO:5) and 3′-UTR (SEQ ID NO:6) regulatory elements for use ingene expression constructs.

All references, including publications, patents, and patentapplications, cited herein are hereby incorporated by reference to theextent they are not inconsistent with the explicit details of thisdisclosure, and are so incorporated to the same extent as if eachreference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention. The followingexamples are provided to illustrate certain particular features and/orembodiments. The examples should not be construed to limit thedisclosure to the particular features or embodiments exemplified.

What is claimed is:
 1. A nucleic acid vector comprising a promoteroperably linked to a heterologous nucleic acid sequence, wherein saidpromoter comprises SEQ ID NO: 5, wherein the nucleic acid vector furthercomprises a 3′ untranslated sequence comprising SEQ ID NO: 6, andwherein the 3′ untranslated sequence is operably linked to saidheterologous nucleic acid sequence.
 2. The nucleic acid vector of claim1, wherein said promoter is less than 3kb in length.
 3. The nucleic acidvector of claim 1, wherein said promoter consists of SEQ ID NO:
 5. 4.The nucleic acid vector of claim 1, wherein said heterologous nucleicacid sequence is a transgene or small RNA.
 5. The nucleic acid vector ofclaim 4, wherein the transgene encodes a selectable marker or a geneproduct conferring insecticidal resistance, herbicide tolerance,nitrogen use efficiency, water use efficiency, or nutritional quality.6. A plant comprising SEQ ID NO: 5 operably linked to a heterologoustransgene, and wherein the plant further comprises a 3′ untranslatedsequence comprising SEQ ID NO: 6, wherein the 3′ untranslated sequenceis operably linked to said transgene.
 7. The plant of claim 6, whereinsaid plant is selected from the group consisting of Zea mays, wheat,rice, sorghum, oats, rye, bananas, sugar cane, soybean, cotton,sunflower, Arabidopsis, tobacco, and canola.
 8. The plant of claim 6,wherein the plant is Zea mays.
 9. The plant of claim 6, wherein thetransgene is inserted into the genome of the plant.